Modulators of the function of FAS receptors and other proteins

ABSTRACT

The present invention provides proteins capable of modulating or mediating the FAS receptor ligand or TNF effect on cells carrying FAS receptor or p55 receptor by binding or interacting with MORT-1 protein, which in turn binds to the intracellular domain of the FAS receptor or to another protein TRADD which binds to the p55 receptor. In addition, peptide inhibitors which interfere with the proteolytic activity of MORT-1-biding proteins having proteolytic activity are provided as well as a method of designing them.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a divisional application of application Ser.No. 08/983,502, filed Apr. 10, 1998, now U.S. Pat. No. 6,399,327, whichis the national stage under 35 U.S.C. §371 of PCT/US96/10521, filed Jun.14, 1996.

FIELD OF THE INVENTION

The present invention is generally in the field of receptors belongingto the TNF/NGF superfamily of receptors and the control of theirbiological functions. The TNF/NGF superfamily of receptors includesreceptors such as the p55 and p75 tumor necrosis factor receptors(TNF-Rs, hereinafter called p55-R and p75-R) and the FAS ligand receptor(also called FAS/APO1 or FAS-R and hereinafter will be called FAS-R) andothers. More specifically, the present invention concerns novel proteinswhich bind to the protein MORT-1 (or FADD), and more specifically, itrelates to one such MORT-1 binding protein, herein designated MACH.

Accordingly, the present invention concerns, in general, new proteinswhich are capable of modulating or mediating the function of MORT-1 orof other proteins which bind to MORT-1 directly or indirectly. Inparticular, the present invention concerns MACH, its preparation anduses thereof, as well as the various novel isoforms of MACH, theirpreparation and uses.

BACKGROUND OF THE RELATED ART

Tumor Necrosis Factor (TNF-α) and Lymphotoxin (TNF-β) (hereinafter, TNF,refers to both TNF-α and TNF-β) are multifunctional pro-inflammatorycytokines formed mainly by mononuclear phagocytes, which have manyeffects on cells (Wallach, D. (1986) In: Interferon 7 (Ion Gresser,ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami(1987)). Both TNF-α and TNF-β initiate their effects by binding tospecific cell surface receptors. Some of the effects are likely to bebeneficial to the organism: they may destroy, for example, tumor cellsor virus infected cells and augment antibacterial activities ofgranulocytes. In this way, TNF contributes to the defense of theorganism against tumors and infectious agents and contributes to therecovery from injury. Thus, TNF can be used as an anti-tumor agent inwhich application it binds to its receptors on the surface of tumorcells and thereby initiates the events leading to the death of the tumorcells. TNF can also be used as an anti-infectious agent.

However, both TNF-α and TNF-β also have deleterious effects. There isevidence that overproduction of TNF-α can play a major pathogenic rolein several diseases. For example, effects of TNF-α, primarily on thevasculature, are known to be a major cause for symptoms of septic shock(Tracey et al., 1986). In some diseases, TNF may cause excessive loss ofweight (cachexia) by suppressing activities of adipocytes and by causinganorexia, and TNF-α was thus called cachetin. It was also described as amediator of the damage to tissues in rheumatic diseases (Beutler andCerami, 1987) and as a major mediator of the damage observed ingraft-versus-host reactions (Piquet et al., 1987). In addition, TNF isknown to be involved in the process of inflammation and in many otherdiseases.

Two distinct, independently expressed, receptors, the p55 and p75TNF-Rs, which bind both TNF-α and TNF-β specifically, initiate and/ormediate the above noted biological effects of TNF. These two receptorshave structurally dissimilar intracellular domains suggesting that theysignal differently (See Hohmann et al., 1989; Engelmann et al., 1990;Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990;Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990).However, the cellular mechanisms, for example, the various proteins andpossibly other factors, which are involved in the intracellularsignaling of the p55 an p75 TNF-Rs have yet to be elucidated. It is thisintracellular signaling, which occurs usually after the binding of theligand, i.e., TNF (α or β), to the receptor, that is responsible for thecommencement of the cascade of reactions that ultimately result in theobserved response of the cell to TNF.

As regards the above-mentioned cytocidal effect of TNF, in most cellsstudied so far, this effect is triggered mainly by the p55 TNF-R.Antibodies against the extracellular domain (ligand binding domain) ofthe p55 TNF-R can themselves trigger the cytocidal effect (see EP412486) which correlates with the effectivity of receptor cross-linkingby the antibodies, believed to be the first step in the generation ofthe intracellular signaling process. Further, mutational studies(Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that thebiological function of the p55 TNF-R depends on the integrity of itsintracellular domain, and accordingly it has been suggested that theinitiation of intracellular signaling leading to the cytocidal effect ofTNF occurs as a consequence of the association of two or moreintracellular domains of the p55 TNF-R. Moreover, TNF (α and β) occursas a homotrimer, and as such, has been suggested to induce intracellularsignaling via the p55 TNF-R by way of its ability to bind to and tocross-link the receptor molecules, i.e., cause receptor aggregation.

Another member of the TNF/NGF superfamily of receptors is the FASreceptor (FAS-R) which has also been called the FAS antigen, acell-surface protein expressed in various tissues and sharing homologywith a number of cell-surface receptors including TNF-R and NGF-R. TheFAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991),and appears to serve as a negative selector of autoreactive T cells,i.e., during maturation of T cells, FAS-R mediates the apoptopic deathof T cells recognizing self-antigens. It has also been found thatmutations in the FAS-R gene (lpr) cause a lymphoproliferation disorderin mice that resembles the human autoimmune disease systemic lupuserythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for theFAS-R appears to be a cell-surface associated molecule carried by,amongst others, killer T cells (or cytotoxic T lymphocytes—CTLS), andhence when such CTLs contact cells carrying FAS-R, they are capable ofinducing apoptopic cell death of the FAS-R-carrying cells. Further, amonoclonal antibody has been prepared that is specific for FAS-R, thismonoclonal antibody being capable of inducing apoptopic cell death incells carrying FAS-R, including mouse cells transformed by cDNA encodinghuman FAS-R (Itoh et al., 1991).

While some of the cytotoxic effects of lymphocytes are mediated byinteraction of a lymphocyte-produced ligand with the widely occurringcell surface receptor FAS-R (CD95), which has the ability to triggercell death, it has also been found that various other normal cells,besides T lymphocytes, express the FAS-R on their surface and can bekilled by the triggering of this receptor. Uncontrolled induction ofsuch a killing process is suspected to contribute to tissue damage incertain diseases, for example, the destruction of liver cells in acutehepatitis. Accordingly, finding ways to restrain the cytotoxic activityof FAS-R may have therapeutic potential.

Conversely, since it has also been found that certain malignant cellsand HIV-infected cells carry the FAS-R on their surface, antibodiesagainst FAS-R, or the FAS-R ligand, may be used to trigger the FAS-Rmediated cytotoxic effects in these cells and thereby provide a meansfor combating such malignant cells or HIV-infected cells (see Itoh etal., 1991). Finding yet other ways for enhancing the cytotoxic activityof FAS-R may therefore also have therapeutic potential.

It has been a long felt need to provide a way for modulating thecellular response to TNF (α or β) and FAS-R ligand. For example, in thepathological situations mentioned above, where TNF or FAS-R ligand isoverexpressed, it is desirable to inhibit the TNF- or FAS-Rligand-induced cytocidal effects, while in other situations, e.g., woundhealing applications, it is desirable to enhance the TNF effect, or inthe case of FAS-R, in tumor cells or HIV-infected cells, it is desirableto enhance the FAS-R mediated effect.

A number of approaches have been made by the laboratory of theapplicants (see for example, European Application Nos. EP 186833, EP308378, EP 398327 and EP 412486) to regulate the deleterious effects ofTNF by inhibiting the binding of TNF to its receptors using anti-TNFantibodies or by using soluble TNF receptors (being essentially thesoluble extracellular domains of the receptors) to compete with thebinding of TNF to the cell surface-bound TNF-Rs. Further, on the basisthat TNF-binding to its receptors is required for the TNF-inducedcellular effects, approaches by the laboratory of of the applicants (seefor example EPO 568925) have been made to modulate the TNF effect bymodulating the activity of the TNF-Rs.

Briefly, EPO 568925 relates to a method of modulating signaltransduction and/or cleavage in TNF-Rs whereby peptides or othermolecules may interact either with the receptor itself or with effectorproteins interacting with the receptor, thus modulating the normalfunction of the TNF-Rs. In EPO 568925, there is described theconstruction and characterization of various mutant p55 TNF-Rs, havingmutations in the extracellular, transmembrane, and intracellular domainsof the p55 TNF-R. In this way, regions within the above domains of thep55 TNF-R were identified as being essential to the functioning of thereceptor, i.e., the binding of the ligand (TNF) and the subsequentsignal transduction and intracellular signaling which ultimately resultsin the observed TNF-effect on the cells. Further, there is alsodescribed a number of approaches to isolate and identify proteins,peptides or other factors which are capable of binding to the variousregions in the above domains of the TNF-R, which proteins, peptides andother factors may be involved in regulating or modulating the activityof the TNF-R. A number of approaches for isolating and cloning the DNAsequences encoding such proteins and peptides; for constructingexpression vectors for the production of these proteins and peptides;and for the preparation of antibodies or fragments thereof whichinteract with the TNF-R or with the above proteins and peptides thatbind various regions of the TNF-R, are also set forth in EPO 568925.However, EPO 568925 does not specify the actual proteins and peptideswhich bind to the intracellular domains of the TNF-Rs (e.g., p55 TNF-R),nor does it describe the yeast two-hybrid approach to isolate andidentify such proteins or peptides which bind to the intracellulardomains of TNF-Rs. Similarly, heretofore there has been no disclosure ofproteins or peptides capable of binding the intracellular domain ofFAS-R.

Thus, when it is desired to inhibit the effect of TNF, or the FAS-Rligand, it would be desirable to decrease the amount or the activity ofTNF-Rs or FAS-R at the cell surface, while an increase in the amount orthe activity of TNF-Rs or FAS-R would be desired when an enhanced TNF orFAS-R ligand effect is sought. To this end the promoters of both the p55TNF-R and the p75 TNF-R have been sequenced, analyzed and a number ofkey sequence motifs have been found that are specific to varioustranscription regulating factors, and as such the expression of theseTNF-Rs can be controlled at their promoter level, i.e., inhibition oftranscription from the promoters for a decrease in the number ofreceptors, and an enhancement of transcription from the promoters for anincrease in the number of receptors (EP 606869 and WO 9531206).Corresponding studies concerning the control of FAS-R at the level ofthe promoter of the FAS-R gene have yet to be reported.

While it is known that the tumor necrosis factor (TNF) receptors, andthe structurally-related receptor FAS-R, trigger in cells, uponstimulation by leukocyte-produced ligands, destructive activities thatlead to their own demise, the mechanisms of this triggering are stilllittle understood. Mutational studies indicate that in FAS-R and the p55TNF receptor (p55-R) signaling for cytotoxicity involve distinct regionswithin their intracellular domains (Brakebusch et al., 1992; Tartagliaet al., 1993; Itoh and Nagata, 1993). These regions (the ‘deathdomains’) have sequence similarity. The ‘death domains’ of both FAS-Rand p55-R tend to self-associate. Their self-association apparentlypromotes that receptor aggregation which is necessary for initiation ofsignaling (see Song et al., 1994; Wallach et al., 1994; Boldin et al.,1995), and at high levels of receptor expression can result intriggering of ligand-independent signaling (Bolding et al., 1995).

Thus, prior to WO 9531544 and the present invention, there have not beenprovided proteins which may regulate the effect of ligands belonging tothe TNF/NGF superfamily, such as the TNF or FAS-R ligand effect oncells, by mediation of the intracellular signaling process, whichsignaling is believed to be governed to a large extent by theintracellular domains (ICs) of the receptors belonging to the TNF/NGFsuperfamily of receptors, such as those of the TNF-Rs, i.e. the p55 andp75 TNF-R intracellular domains (p55IC and p75IC, respectively), as wellas the FAS-IC.

Some of the cytotoxic effects of lymphocytes are mediated by interactionof a lymphocyte-produced ligand with FAS-R (CD-95), a widely occurringcell surface receptor which has the ability to trigger cell death (seeNagata and Golstein, 1995). Cell killing by mononuclear phagocytesinvolves a ligand-receptor couple, TNF and its receptor p55-R (CD120),that is structurally related to FAS-R and its ligand (see alsoVandenabeele et al., 1995). Like other receptor-induced effects, celldeath induction by the TNF receptors and FAS-R occurs via a series ofprotein-protein interactions, leading from ligand-receptor binding tothe eventual activation of enzymatic effector functions, which in thecase of these particular receptors results in cell death. Previousstudies have elucidated non-enzymatic protein-protein interactions thatinitiate signaing for cell death: binding of trimeric TNF or the FAS-Rligand molecules to the receptors, the resulting interactions of theirintracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993;Itoh and Nagata, 1993) augmented by a propensity of the death-domainmotifs to self-associate, (Boldin et al., 1995a), and induced binding oftwo cytoplasmic proteins (which can also bind to each other) to thereceptors' intracellular domains—MORT-1 (or FADD) to FAS-R (Boldin etal., 1995b; Chinnaiyan et al., 1995; Kischkel et al., 1995) and TRADD top55-R (Hsu et al., 1995; Hsu et al., 1996).

Three proteins that bind to the intracellular domain of FAS-R and p55-Rat the “death domain” region involved in cell-death induction by thereceptors through hetero-association of homologous regions and thatindependently are also capable of triggering cell death were identifiedby the yeast two-hybrid screening procedure. One of these is theprotein, MORT-1 (Boldin et al. 1995b) also known as FADD (Chinnaiyan etal., 1995), that binds specifically to FAS-R. A second one, TRADD (seealso Hsu et al., 1995, 1996), binds to p55-R, and the third, RIP (seealso Stanger et al., 1995), binds to both FAS-R and p55-R. Besides theirbinding to FAS-R and p55-R, these proteins are also capable of bindingto each other, which provides for a functional “cross-talk” betweenFAS-R and p55-R. These bindings occur through a conserved sequencemotif, the “death domain module” common to the receptors and theirassociated proteins. Furthermore, although in the yeast two-hybrid testMORT-1 was shown to bind spontaneously to FAS-R, in mammalian cells thisbinding takes place only after stimulation of the receptor, suggestingthat MORT-1 participates in the initiating events of FAS-R signaling.MORT-1 does not contain any sequence motif characteristic of enzymaticactivity, and therefore, its ability to trigger cell death seems not toinvolve an intrinsic activity of MORT-1 itself, but rather, activationof some other protein(s) that bind MORT-1 and act further downstream inthe signaling cascade. Cellular expression of MORT-1 mutants lacking theN-terminal part of the molecule has been shown to block cytotoxicityinduction by FAS/APO1 (FAS-R) or p55-R (Hsu et al., 1996; Chinnaiyan etal., 1996), indicating that this N-terminal region transmits thesignaling for the cytocidal effect of both receptors throughprotein-protein interactions.

Recent studies have implicated a group of cytoplasmic thiol proteaseswhich are structurally related to the Caenorhabditis elegans proteaseCED3 and to the mammalian interleukin-1β converting enzyme (ICE) in theonset of various physiological cell death processes (reviewed in Kumar,1995 and Henkart, 1996). There have also been some indications thatprotease(s) of this family may take part in the cell-cytotoxicityinduced by FAS-R and TNF-Rs. Specific peptide inhibitors of theproteases and two virus-encoded proteins that block their function, thecowpox protein crmA and the Baculovirus p35 protein, were found toprovide protection to cells against this cell-cytotoxicity (Enari etal., 1995; Los etal., 1995; Tewari et al., 1995; Xue et al., 1995;Beidler et al., 1995). Rapid cleavage of certain specific cellularproteins, apparently mediated by protease(s) of the CED3/ICE family, wasobserved in cells shortly after stimulation of FAS-R or TNF-Rs.Heretofore, no information has been presented as to the identity of thespecific CED3/ICE-related protease(s) involved, nor of the mechanisms ofactivation of these protease(s) by the receptors.

SUMMARY OF THE INVENTION

It is an object of the invention to provide novel proteins, includingall isoforms, analogs, fragments or derivatives thereof, which arecapable of binding to MORT-1, which itself binds to the intracellulardomain of the FAS-R, which novel proteins affect the intracellularsignaling process initiated by the binding of FAS ligand to itsreceptor.

Another object of the invention is to provide antagonists (e.g.,antibodies, peptides, organic compounds, or even some isoforms) to theabove novel proteins, analogs, fragments and derivatives thereof, whichmay be used to inhibit the signaling process, or, more specifically, thecell-cytotoxicity, when desired.

A further object of the invention is to use the above novel proteins,analogs, fragments and derivatives thereof, to isolate and characterizeadditional proteins or factors, which may be involved in regulation ofreceptor activity, e.g., other proteases which cleave the novel proteinsto render them biologically active, and/or to isolate and identify otherreceptors further upstream in the signaling process to which these novelproteins, analogs, fragments and derivatives bind (e.g., other FAS-Rs orrelated receptors), and hence, in whose function they are also involved.

A still further object of the invention is to provide inhibitors whichcan be introduced into cells to bind or interact with the MACH proteasesand inhibit their proteolytic activity.

Moreover, it is an object of the present invention to use theabove-mentioned novel proteins, and analogs, fragments and derivativesthereof as antigens for the preparation of polyclonal and/or monoclonalantibodies thereto. The antibodies, in turn, may be used, for example,for the purification of the new proteins from different sources, such ascell extracts or transformed cell lines.

Furthermore, these antibodies may be used for diagnostic purposes, e.g.,for identifying disorders related to abnormal functioning of cellulareffects mediated by the FAS-R or other related receptors.

A further object of the invention is to provide pharmaceuticalcompositions comprising the above novel proteins, or analogs, fragmentsor derivatives thereof, as well as pharmaceutical compositionscomprising the above noted antibodies or other antagonists.

In accordance with the present invention, a novel protein, MACH, whichis capable of binding to, or interacting with, MORT-1, which itselfbinds to the intracellular domain of the FAS-R was discovered. MACHprobably functions as an effector component of the cell-death pathwayinitiated by the binding of FAS ligand to FAS-R at the cell surface, andthis by virtue of the fact that at least some of the isoforms of MACHappear to be active intracellular proteases. Proteases of the CED3/ICEfamily have been implicated in the apoptopic process triggered by FAS-R.MORT-1 (or FADD) binds to the intracellular domain of FAS-R uponactivation of this receptor and the novel MACH proteins of the presentinvention bind to MORT-1. The MACH protein, cloned and characterized inaccordance with the present invention, actually exists in multipleisoforms, some of which isoforms have a CED3/ICE homology region whichhas proteolytic activity (proteolytic domain), and causes the death ofcells when expressed in the cells. Thus, activation of this novelCED3/ICE homolog (i.e., the various MACH isoforms having the proteolyticdomain) by FAS-R (via MORT-1 interaction) appears to constitute aneffector component of the FAS-R-mediated cell-death pathway.

Moreover, MACH also appears to function as an effector component of thecell-death pathway initiated by the binding of TNF to p55-R at the cellsurface, this by way of indirect mechanism of MORT-1 binding to TRADD, aprotein which binds to the intracellular domain of p55-R (Hsu et al.,1995), followed by or together with MACH binding to MORT-1, with theactivation of MACH into an active protease involved in effecting celldeath.

It should also be noted that while MACH, in particular, the MACHα1isoform, displays all of the sequence features critical of the functionof the CED3/ICE proteases, it does, however, have some distinctivesequence features of its own which may endow it with a unique andpossibly tissue/cell specific mode of action.

MORT-1 (for ‘Mediator of Receptor Toxicity’, Boldin et al., 1995b),previously designated HF1, is capable of binding to the intracellulardomain of the FAS-R. This FAS-IC-binding protein appear to act as amediator or modulator of the FAS-R ligand effect on cells by way ofmediating or modulating the intracellular signaling process whichusually occurs following the binding of the FAS-R ligand at the cellsurface. In addition to its FAS-IC-binding specificity, MORT-1 was shownto have other characteristics (see Example 1), for example, it has aregion homologous to the “death domain” (DD) regions of the p55-TNF-Rand FAS-R (p55-DD and FAS-DD), and thereby is also capable ofself-association. MORT-1 is also capable of activating cell cytotoxicityon its own, an activity possibly related to its self-associationcapability. It has now also been found that co-expression of the regionin MORT-1 (HF1) that contains the “death domain” homology sequence(MORT-DD, present in the C-terminal part of MORT-1) strongly interfereswith FAS-induced cell death, as would be expected from its ability tobind to the “death domain” of the FAS-IC. Further, in the sameexperimental conditions, it was found that co-expression of the part ofMORT-1 that does not contain the MORT-DD region (the N-terminal part ofMORT-1, amino acids 1-117, “MORT-1 head”) resulted in no interference ofthe FAS-induced cell death and, if at all, a somewhat enhancedFAS-induced cell cytotoxicity.

Accordingly, it is likely that MORT-1 also binds to other proteinsinvolved in the intracellular signaling process. These MORT-1-bindingproteins may therefore also act as indirect mediators or modulators ofthe FAS-R ligand effect on cells by way of mediating or modulating theactivity of MORT-1; or these MORT-1-binding proteins may act directly asmediators or modulators of the MORT-1-associated intracellular signalingprocess by way of mediating or modulating the activity of MORT-1, which,as noted above, has an apparently independent ability to activate cellcytotoxicity. These MORT-1-binding proteins may also be used in any ofthe standard screening procedures to isolate, identify and characterizeadditional proteins, peptides, factors, antibodies, etc., which may beinvolved in the MORT-1-associated or FAS-R-associated signaling processor may be elements of other intracellular signaling processes. SuchMORT-1-binding proteins have been isolated and are described herein (seeExample 2 and Example 3). One of these MORT-1-binding proteins, hereindesignated MACH, was initially cloned, sequenced, and partiallycharacterized as having the following properties: The MACH cDNA encodesthe ORF-B open-reading frame; MACH binds to MORT-1 in a very strong andspecific manner; the MACH binding site in MORT-1 occurs upstream of theMORT-1 “death domain” motif; the ORF-B region of MACH is theMORT-1-interacting part thereof; and MACH is capable of self-associationand of inducing cell-cytotoxicity on its own.

In accordance with the present invention, it has now been shown asmentioned above, that MACH actually exists in a number of isoforms.Moreover, the MACH ORF-B noted above is in fact one of the MACH isoformsdesignated herein as MACHβ1 (see below).

Accordingly, the present invention provides a DNA sequence encoding aprotein, analogs or fragments thereof, capable of binding to orinteracting with MORT-1, said protein, analogs or fragments thereofbeing capable of mediating the intracellular effect mediated by theFAS-R or p55-TNF-R.

In particular, the present invention provides a DNA sequence selectedfrom the group consisting of:

(a) a cDNA sequence derived from the coding region of a native MORT-1binding protein;

(b) DNA sequences capable of hybridization to a sequence of (a) undermoderately stringent conditions and which encode a biologically activeMORT-1 binding protein; and

(c) DNA sequences which are degenerate as a result of the genetic codeto the DNA sequences defined in (a) and (b) and which encode abiologically active MORT-1 binding protein.

Another specific embodiment of the above DNA sequence of the inventionis a DNA sequence comprising at least part of the sequence encoding atleast one isoform of the MACH protein selected from the hereindesignated MACH isoforms MACHα1, MACHα2, MACHα3, MACHβ2, MACHβ1, MACHβ3,MACHβ4 and MACHβ5.

Other specific embodiments of the DNA sequence of the invention as notedabove are DNA sequences encoding:

(a) a MACH isoform selected from MACHα1, MACHβ1 and MACHβ3 having anamino acid sequence set forth in SEQ ID NOs:7, 5 and 8 respectively, andanalogs and fragments of any one thereof;

(b) MACHα1 having the amino acid sequence set forth in SEQ ID NO:7, andanalogs and fragments thereof;

(c) MACHβ1 having the amino acid sequence set forth in SEQ ID NO:5, andanalogs and fragments thereof;

(d) MACHβ3 having the amino acid sequence set forth in SEQ ID NO:8, andanalogs and fragments thereof.

In the present invention provides MORT-1-binding proteins, and analogs,fragments or derivatives thereof encoded by any of the above sequencesof the invention, said proteins, analogs, fragments and derivativesbeing capable of binding to or interacting with MORT-1 and mediating theintracellular effect mediated by the FAS-R or p55 TNF-R.

A specific embodiment of the invention is the MORT-1-binding protein,analogs fragments and derivatives thereof, which are selected from asleast one isoform of MACH of the group comprising MACHα1, MACHα2,MACHα3, MACHβ1, MACHβ2, MACHβ3, MACHβ4 and MACHβ5 which have at leastpart of the amino acid sequences thereof.

Also provided by the present invention are vectors encoding the aboveMORT-1-binding protein, and analogs, fragments or derivatives of theinvention, which contain the above DNA sequence of the invention, thesevectors being capable of being expressed in suitable eukaryotic orprokaryotic host cells; transformed eukaryotic or prokaryotic host cellscontaining such vectors; and a method for producing the MORT-1-bindingprotein, or analogs, fragments or derivatives of the invention bygrowing such transformed host cells under conditions suitable for theexpression of said protein, analogs, fragments or derivatives, effectingpost-translational modifications of said protein as necessary forobtaining said protein and extracting said expressed protein, analogs,fragments or derivatives from the culture medium of said transformedcells or from cell extracts of said transformed cells. The abovedefinitions are intended to include all isoforms of the MACH protein.

In another aspect, the present invention also provides antibodies oractive derivatives or fragments thereof specific the MORT-1-bindingprotein, and analogs, fragments and derivatives thereof, of theinvention.

By yet another aspect of the invention, there are provided various usesof the above DNA sequences or the proteins which they encode, accordingto the invention, which uses include amongst others:

(i) A method for the modulation of the FAS-R ligand or TNF effect oncells carrying a FAS-R or p55-R, comprising treating said cells with oneor more MORT-1-binding proteins, analogs, fragments or derivatives ofthe invention, capable of binding to MORT-1, which binds to theintracellular domain of FAS-R, or capable of binding to MORT-1 whichbinds to TRADD which binds to the intracellular domain of p55-R, andthereby being capable of modulating/mediating the activity of said FAS-Ror p55 TNF-R, wherein said treating of said cells comprises introducinginto said cells said one or more proteins, analogs, fragments orderivatives in a form suitable for intracellular introduction thereof,or introducing into said cells a DNA sequence encoding said one or moreproteins, analogs, fragments or derivatives in the form of a suitablevector carrying said sequence, said vector being capable of effectingthe insertion of said sequence into said cells in a way that saidsequence is expressed in said cells.

(ii) A method for the modulation of the FAS-R ligand or TNF effect oncells according to (i) above, wherein said treating of cells comprisesintroducing into said cells said MORT-1-binding protein, or analogs,fragments or derivatives thereof, in a form suitable for intracellularintroduction, or introducing into said cells a DNA sequence encodingsaid MORT-1-binding protein, or analogs, fragments or derivatives in theform of a suitable vector carrying said sequence, said vector beingcapable of effecting the insertion of said sequence into said cells in away that said sequence is expressed in said cells.

(iii) A method as in (ii) above wherein said treating of said cells isby transfection of said cells with a recombinant animal virus vectorcomprising the steps of

(a) constructing a recombinant animal virus vector carrying a sequenceencoding a viral surface protein (ligand) that is capable of binding toa specific cell surface receptor on the surface of a FAS-R- orp55-R-carrying cell and a second sequence encoding a protein selectedfrom MORT-1-binding protein, and analogs, fragments and derivativesthereof, that when expressed in said cells is capable ofmodulating/mediating the activity of said FAS-R or p55-R; and

(b) infecting said cells with said vector of (a).

(iv) A method for modulating the FAS-R ligand or TNF effect on cellscarrying a FAS-R or a p55-R comprising treating said cells withantibodies or active fragments or derivatives thereof, according to theinvention, said treating being by application of a suitable compositioncontaining said antibodies, active fragments or derivatives thereof tosaid cells, wherein when the MORT-1-binding protein, or portions thereofof said cells are exposed on the extracellular surface, said compositionis formulated for extracellular application, and when saidMORT-1-binding proteins are intracellular, said composition isformulated for intracellular application.

(v) A method for modulating the FAS-R ligand or TNF effect on cellscarrying a FAS-R or p55-R comprising treating said cells with anoligonucleotide sequence encoding an antisense sequence of at least partof the MORT-1-binding protein sequence of the invention, saidoligonucleotide sequence being capable of blocking the expression of theMORT-1-binding protein.

(vi) A method as in (ii) above for treating tumor cells or HIV-infectedcells or other diseased cells, comprising:

(a) constructing a recombinant animal virus vector carrying a sequenceencoding a viral surface protein capable of binding to a specific tumorcell surface receptor or HIV-infected cell surface receptor or receptorcarried by other diseased cells and a sequence encoding a proteinselected from MORT-1-binding protein, analogs, fragments and derivativesof the invention, that when expressed in said tumor, HIV-infected, orother diseased cell is capable of killing said cell; and

(b) infecting said tumor or HIV-infected cells or other diseased cellswith said vector of (a).

(vii) A method for modulating the FAS-R ligand or TNF effect on cellscomprising applying the ribozyme procedure in which a vector encoding aribozyme sequence capable of interacting with a cellular mRNA sequenceencoding a MORT-1-binding protein according to the invention, isintroduced into said cells in a form that permits expression of saidribozyme sequence in said cells, and wherein when said ribozyme sequenceis expressed in said cells it interacts with said cellular mRNA sequenceand cleaves said mRNA sequence resulting in the inhibition of expressionof said MORT-1-binding protein in said cells.

(viii) A method selected from the method according to the invention,wherein said MORT-1-binding protein encoding sequence comprises at leastone of the MACH isoforms, analogs, fragments and derivatives of anythereof according to the invention which are capable of bindingspecifically to MORT-1 which in turn binds specifically to FAS-IC, orwhich are capable of binding to MORT-1 which in turn binds to TRADD andwhich in turn binds to the p55-IC.

(ix) A method for isolating and identifying proteins, according to theinvention, capable of binding to the MORT-1 protein, comprising applyingthe yeast two-hybrid procedure in which a sequence encoding said MORT-1protein is carried by one hybrid vector and sequence from a cDNA orgenomic DNA library is carried by the second hybrid vector, the vectorsthen being used to transform yeast host cells and the positivetransformed cells being isolated, followed by extraction of the saidsecond hybrid vector to obtain a sequence encoding a protein which bindsto said MORT-1 protein, said protein being the MORT-1-binding proteins.

(x) A method according to any one of (i)-(ix) above wherein saidMORT-1-binding protein is the MACH isoform herein designated MACHα1,analogs, fragments and derivatives thereof.

(xi) A method according to any one of (i)-(ix) above wherein saidMORT-1-binding protein is the MACH isoform herein designated MACHβ1,analogs, fragments and derivatives thereof.

(xii) A method according to any one of (i)-(ix) above wherein saidMORT-1-binding protein is the MACH isoform herein designated MACHβ3,analogs, fragments and derivatives thereof.

The present invention also provides a pharmaceutical composition for themodulation of the FAS-R ligand- or TNF-effect on cells comprising, asactive ingredient any one of the following:

(i) a MORT-1-binding protein according to the invention, andbiologically active fragments, analogs, derivatives or mixtures thereof;

(ii) a recombinant animal virus vector encoding a protein capable ofbinding a cell surface receptor and encoding a MORT-1-binding protein orbiologically active fragments or analogs, according to the invention;

(iii) an oligonucleotide sequence encoding an anti-sense sequence of theMORT-1-binding protein sequence according to the invention, wherein saidoligonucleotide may be the second sequence of the recombinant animalvirus vector of (ii) above.

The present invention also provides:

I. a method for the modulation of the MORT-1-induced effect orMORT-1-binding protein-induced effect on cells comprising treating saidcells in accordance with a method of any one of (i)-(xi) above, withMORT-1-binding proteins, analogs, fragments or derivatives thereof orwith sequences encoding MORT-1-binding proteins, analogs or fragmentsthereof, said treatment resulting in the enhancement or inhibition ofsaid MORT-1-mediated effect, and thereby also of the FAS-R orp55-R-mediated effect.

II. a method as above wherein said MORT-1-binding protein, analog,fragment or derivative thereof is that part of the MORT-1-bindingprotein which is specifically involved in binding to MORT-1 orMORT-1-binding protein itself, or said MORT-1-binding protein sequenceencodes that part of MORT-1-binding protein which is specificallyinvolved in binding to MORT-1 or the MORT-1-binding protein itself.

III. A method as above wherein said MORT-1-binding protein is any one ofthe MACH isoforms selected from MACHα1, MACHβ1, and MACHβ3, said MACHisoforms capable of enhancing the MORT-1-associated effect on cells andthereby also of enhancing the FAS-R- or p55-R-associated effect oncells.

As arises from all the above-mentioned, as well as from the detaileddescription hereinbelow, MACH may be used in a MORT-1 independentfashion to treat cells or tissues. Isolation of the MORT-1-bindingproteins, their identification and characterization may be carried outby any of the standard screening techniques used for isolating andidentifying proteins, for example, the yeast two-hybrid method, affinitychromatography methods, and any of the other well-known standardprocedures used for this purpose.

Other aspects and embodiments of the present invention are also providedas arising from the following detailed description of the invention.

It should be noted that, where used throughout, the following terms:“Modulation of the FAS-ligand or TNF effect on cells”; and “Modulationof the MORT-1 or MORT-1-binding protein effect on cells” are understoodto encompass in vitro as well as in vivo treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the interaction of MORT-1 with FAS-IC and theself-association of MORT-1 within transformed yeasts as assessed by atwo-hybrid β-galactosidase expression test.

FIG. 2 depicts schematically the preliminary nucleotide (SEQ ID NO:1)and deduced amino acid sequence (SEQ ID NO:2) of MORT-1 (HF1) in whichthe ‘death domain’ is underlined as is a possible translation startsite, i.e., the underlined methionine residue at position 49 (bold,underlined M). The asterisk indicates the translation stop codon(nucleotides 769-771). At the beginning and in the middle of each lineare provided two numerals depicting the relative positions of thenucleotides and amino acids of the sequence with respect to the start ofthe sequence (5′ end), in which the first numeral denotes the nucleotideand the second numeral denoted the amino acid.

FIG. 3 is a preliminary partial nucleotide sequence (SEQ ID NO:3)encoding a MORT-1 binding protein as obtained from a cDNA clone.

FIGS. 4A-C depict schematically the MACH cDNA and its encoded protein,wherein in FIG. 4A there is shown the structure of the MACH cDNA whichencodes two MACH open-reading frames (ORF-A and ORF-B), the hatched areaof ORF-B indicating the region thereof having homology with the MORT-1protein; in FIG. 4B, there is shown the deduced amino acid sequence (SEQID NO:5) for the MACH ORF-B region, the underlined amino acid residuesbeing the region having homology with MORT-1 (corresponding to thehatched region of FIG. 4A); and in FIG. 4C, there is shown thenucleotide sequence (SEQ ID NO:4) of the entire MACH cDNA molecule(designated MACHβ1).

FIG. 5 depicts the results illustrating the interaction of MORT-1 andMACH within transfected yeast cells.

FIG. 6 depicts graphically the ligand-independent triggering ofcytocidal effects in HeLa cells transfected with tetracycline-controlledexpression vectors encoding MACH, as compared to the effects in thesecells transfected with such vectors encoding other proteins such asluciferase (luc, negative control), FAS-IC, MORT-1, and cellsco-transfected with vectors encoding MORT-1 and MACH.

FIGS. 7A and 7B show the amino acid sequence (SEQ ID NO:5) of MACHβ1(FIG. 7A). FIG. 7B shows the sequence homology of the MORT module inMACHβ1, MORT-1 and PEA-15 (SEQ ID NO:6).

FIG. 8 is a diagrammatic representation of the receptor and targetinteractions participating in induction of cell-death by Fas/APO1 andp55, the death domain module being indicated by stripes, the MORT modulebeing indicated in gray and the CED3/ICE region being indicated inblack.

FIGS. 9A-C depict the results illustrating the in vitro interaction ofMACHβ1 and its deletion mutants with MORT-1. FIG. 9A shows theassessment of the expression of the proteins and their molecular sizesby immunoprecipitation from cell lysates using anti-FLAG antibody. FIG.9B shows affinity binding of the proteins to GST-MORT-1, adsorbed toglutathione-agarose beads (or, as a control, to GST or GST-fused to theintracellular domain of Fas-APO1). FIG. 9C shows the results of theimmunoprecipitations of the various MORT-1 and MACH fusion constructsusing the various specific antibodies.

FIG. 10 is a diagrammatic representation of the various MACH isoforms.

FIG. 11 is a schematic colinear amino acid sequence alignment of theMACH isoforms, MACHα1 (SEQ ID NO:7), MACHβ1 (SEQ ID NO:5), and MACHβ3(SEQ ID NO:8) and the various known members of the CED3/ICE proteasefamily, CED-3 (SEQ ID NO:9), Ich-11/Nedd2 (SEQ ID NO:10), ICE_(rel)III(SEQ ID NO:11), Tx/Ich2/ICE_(rel)II (SEQ ID NO:12), ICE (SEQ ID NO:13),CPP-32 (SEQ ID NO:30), Mcn2α (SEQ ID NO:31). Amino acid residues arenumbered both to the left and to the right of each sequence. Dottedlines; gaps in the sequence to allow optimal alignment. Amino acids thatare identical in at least three of the members of the CED3/ICE proteasefamily show are boxed. The MORT modules upstream to CED3/ICE homologyregion are boxed. Sites of C-terminal deletions employed in this studyare denoted by broken lines. The four amino acid blocks downstream tothe MORT module region, which vary among the various MACH isoforms(blocks 1-4) are denoted by overlinings. Within the CED3/ICE homologyregion, amino acids aligning with residues within ICE implicated incatalytic activity by X-ray crystal analysis are denoted as follows: Theresidues putatively involved in catalysis, corresponding to His₂₃₇,Gly₂₃₈ and Cys₂₈₅ in ICE are marked by closed circles below thealignment (). The residues constituting the binding pocket for thecarboxylate side chain of the P1 Asp, corresponding to Arg₁₇₉,corresponding to Arg₁₇₉, Gln₂₈₃, Arg341 and Ser347 in ICE, are marked byopen circles below the alignment (◯). The Ala residues upstream to theresidues corresponding to Cys285 in ICE, and the Arg and Gly residuesdownstream to this Cys, which are conserved in all previously describedproteases of the CED3/ICE family. Residues proximal to P₁-P₄ residues ofthe substrate are marked by triangles below the alignment (Δ). Known andpreviously suspected Asp-X cleavage sites and potential sites ofcleavage found at similar locations in MACH are boxed. Arrows indicatethe N- and C-terminal ends of the p20 and p10 subunits of ICE and of thep17 and p12 subunits of CPP32. The C-termini of the proteins are denotedby asterisks (*).

FIGS. 12A-F depict the results illustrating the protease activity ofMACHα1 at 15 min. (FIG. 12A), 30 min. (FIG. 12B), 60 min. (FIG. 12C), 90min. (FIG. 12D), 180 min. (FIG. 12E). FIG. 12F shows the proteolyticactivity over time at a specific concentration of substrate.

FIGS. 13A and 13B show the protease activity of the CED3/ICE homologyregion in MACHα.A, Kinetics of cleaage of the PARP-sequence-derivedfluorogenic substrate, Ac-DEVD-AMC (50 μM), by extracts of E. coliexpressing a GST-fusion protein of the CED3/ICE homology region inMACHα1 (Ser₂₁₇ through the C-terminus of the protein (▪) as compared tothe lack of cleavage by extracts of bacteria expressing GST-fusionproducts of the full-length MACHα1 (◯), or of the CED3/ICE homologyregion in which Cys360 was replaced by Ser (∇), or by extracts ofbacteria expressing GST-fusion products of either of the two potentialproteolytic products of the CED3/ICE homology region (Ser₂₁₇ thoughAsp₃₇₃ (Δ) and Ser₃₇₅ through Asp₄₇₉, the C-terminus of the protein(▴)).B, Substrate-concentration dependence of the cleavage ofAc-DEVD-AMC. The substrate was incubated for 180 min with extracts ofbacteria expressing the GST-fusion product of the MACHα1 CED3/ICEhomology region (▪). Cleavage of this substrate by the extracts wasinhibited in the presence of iodoacetic acid (5 mM, □). Ac-YVAD-AMC, afluorogenic substrate corresponding to an ICE cleavage site in theIL-l1β precursor, was not cleaved ().

FIGS. 14A-D show cell death mediated by MACHα1 and MACHα2.

FIG. 15 depict graphically cell death mediated by MACHα1 and MACHα2.

FIGS. 16A-D show the morphology of cells in which cell death was inducedor blocked.

FIG. 17 graphically shows that MACHα molecules that contain anon-functional CED3/ICE region block cell death induction by p55-R.

FIG. 18 shows that MACHα molecules that contain a non-functionalCED3/ICE region block cell death induction by FAS/APO1.

FIG. 19 shows death of HeLa cells that transiently express FAS/APO1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to novel MORT-1-bindingproteins which are capable of binding to or interacting with MORT-1 andthereby of binding to the intracellular domain of the FAS-R receptor, towhich MORT-1 binds, or of binding to the intracellular domain of the p55TNF-R, to which the protein TRADD (see Example 2 and as noted above)binds and to which TRADD protein MORT-1 binds. Hence, the MORT-1 bindingproteins of the present invention are considered as mediators ormodulators of FAS-R, having a role in, for example, the signalingprocess that is initiated by the binding of FAS ligand to FAS-R, andlikewise also having a role in the signaling process that is initiatedby the binding of TNF to p55-R. Of the MORT-1-binding proteins of thepresent invention are included the newly discovered MACH isoforms, theamino acid and DNA sequences of which are new sequences not appearing inthe ‘GENBANK’ or ‘PROTEIN BANK’ data banks of DNA or amino acidsequences.

More particularly, in accordance with the present invention, severalmammalian homologs of the nematode protease, CED3 have been disclosed.These have been designated as MACH isoforms (MACHα and MACHβ isoforms)which, although being closely related, do however display somedifferences of structure and of substrate specificity, and as such mayserve somewhat different functions in mammalian cells. Indeed, twodifferent activities of the proteases are known. The main role of ICEseems to be the processing of the IL-1β precursor, while CED3 has beenclearly shown to serve as an effector of programmed cell death. Thislatter role also appears to be the role of at least some of themammalian homologs (some MACH isoforms). The amino acid sequence ofMACHα1 shows closest resemblance to CPP32, the closest known mammalianhomolog of CED3. The substrate specificity of MACH is also similar tothat of CPP32, except that MACHα1 seems to have a more restrictedsubstrate specificity than that of CPP32. CPP32 cleaves preferentiallythe substrate peptide corresponding to a cleavage site in poly (ADPribose) polymerase (PARP), yet also has some proteolytic activityagainst the peptide corresponding to the ICE cleavage site in the IL-1βprecursor. MACHα1 seems, however, to be solely capable of cleaving thePARP-derived sequence. These relationships of MACHα1 to CPP32 and CED3,and its dissimilarities to ICE, are consistent with the idea that MACHα1serves, similarly to CED3, as regulator of cell death. MACHα1 displays,though, some sequence features which distinguish it from CED3 and fromCPP32, as well as from all other members of the CED3/ICE family. The Cterminal part of MACHα1, upstream to its CED3/ICE homology region, showsno resemblance at all to the upstream region of any of the otherhomologs. There are also some unique sequence features to the CED3/ICEhomology region of the protein. These differences suggest that MACHα1belongs to a distinct evolutionary branch of the family and that itscontribution to cell death somewhat differs from that of the previouslydescribed CED3/ICE homologs.

One important difference may concern the way in which the function ofthe protease is regulated. Being involved both in developmentallyrelated cell death processes and in receptor-induced immune cytolysis,the cleavage of proteins by proteases of the CED3/ICE family should beamenable to regulation both by signals that are formed within the celland by signals emanating from cell surface receptors. In developmentalcell death processes, the activation of such proteases seems to involvemechanisms that affect gene expression, resulting in enhanced synthesisof the proteases, as well as in decreased synthesis of proteins likeBCL-2, that antagonize their apoptopic effect. This is clearly not thecase, however, for the cytotoxicity triggered by FAS-R or the TNFreceptors. Cells can be killed by TNF or the FAS-R ligand even whentheir protein synthesis activity is fully blocked (they are in factkilled more effectively then) and remain stimulus-dependent under theseconditions. Activation of proteases of the CED3/ICE family by the TNFreceptors and FAS-R may thus occur by a mechanism which isprotein-synthesis-independent. The unique sequence properties of MACHα1may allow it to take part in such a mechanism.

To applicants' knowledge, no other protease has so far been found toassociate, either directly or through an adapter protein, with theintracellular domain of a cell surface receptor. Yet by inference fromthe way of action of receptor-associated proteins that have otherenzymatic activities, it seems plausible that the binding of MACHα1 toMORT1 allows stimulation of the MACHα1 protease-activity upon triggeringof FAS-R. It may also allow activation of the protease by the p55-R,through the binding of MORT1 to TRADD, which binds to p55-R.

Other members of the CED3/ICE family were found to exhibit full activityonly after proteolytic processing, which occurs either by theirself-cleavage or by effects of other proteases of this family (reviewedin Kumar, 1995; Henkart, 1996). The cytotoxic effect resulting fromco-expression of the two major potential self-cleavage products ofMACHα1, as opposed to the lack of cytotoxicity in cells that express thefull-length CED3/ICE homology region, is consistent with the possibilitythat also MACHα1 gains full activity only after its processing. Theenzymatic activity observed in lysates of bacteria that express the fulllength region apparently reflect self processing of the protein producedunder these conditions or processing by some bacterial proteases. Inwhat way this processing occurs within the mammalian cell, and how itcan be brought about by triggering of FAS-R and p55-R, is not known, noris it clear yet what relative contribution the protease activity ofMACHα1 makes to the FAS-R- and TNF-induced cytotoxicity. Evaluation ofthis contribution is complicated by the fact that also expression ofMACHβ1, which lacks the CED3/ICE homology region, results in markedcytotoxicity. Presumably, this cytotoxicity reflects the ability ofMACHβ1 to bind to MACHα1. Due to this ability, transfected MACHmolecules may impose, upon aggregation, a conformational change in theMACHα1 molecules that are endogenous to the transfected cell. Such amechanism may well account also for the cytotoxicity observed whenmolecules that act upstream to MACH, (MORT1, TRADD or the death domainsof either the p55-R or FAS-R) are over-expressed in cells. At themoment, however, one cannot exclude that the cytotoxicity observed uponinduced expression of MACH or of molecules that act upstream to itreflect, besides the proteolytic activity of the CED3/ICE homologyregion in MACH, also activation of some of the other mechanisms believedto take part in the FAS-R and p55-R cytotoxic effect (for example,activation of the neutral or acid sphingomyelinase). One also cannotexclude that the proteolytic activity of the CED3/ICE homology regionserves other functions besides cytotoxicity induction. A clearer idea ofthe function of MACHα1 should be gained by identification of theendogenous substrate proteins that are cleaved upon activation ofMACHα1. Finding ways to ablate the activity of MACHα1 at will, forexample by expression of inhibitory molecules, will also contribute tounderstanding of the function of this protein, and serve as a way forregulating its activity when desired.

There may well exist within cells that express MACHα1 natural inhibitorsof the protease encompassed in this protein. Existence of alternativelyspliced isoforms for some of the other members of the CED3/ICE familyhas been shown to constitute a way of physiological restriction of thefunction of these proteases. Some of the isoforms of these otherproteases were reported to act as natural inhibitors of the full-lengthisoforms, apparently by forming inactive heterodimers, with them. Thismay well be the case also for some isoforms of MACH, for example,MACHα3, in which the potential N-terminal cleavage site is missing andMACHα1 mutants whose CED3/ICE homology region is deficient. Expressionof such inhibitory isoforms may constitute a mechanism of cellularself-protection against the FAS-R and TNF cytotoxicity. The wideheterogeneity of MACH isoforms, which greatly exceeds the heterogeneityobserved for any of the other proteases of the CED3/ICE family, mayallow a particularly refined tuning of the function of the active formof this protein. It seems also possible that some of the MACH isoformsserve other functions. The ability of MACHβ1 to bind both to MORT1 andto MACHα1 raises the possibility that some of these isoforms, andperhaps also other MACH isoforms, do not have an inhibitory but ratheran enhancing effect on the function of the enzymatically activeisoforms. It seems also possible that some isoforms do not serve a rolerelated to cytotoxicity, but rather act as docking sites for moleculesthat are involved in other, non-cytotoxic, effects of FAS-R and TNF.

Due to the unique ability of FAS-R and the TNF receptors to cause celldeath, as well as the ability of the TNF receptors to trigger variousother tissue-damaging activities, aberration of the function of thesereceptors can be particularly deleterious to the organism. Indeed,both-excessive and deficient function of these receptors have been shownto contribute to the pathological manifestations of various diseases.Identifying molecules that take part in the signaling activity of thesereceptors, and finding ways to modulate the function of these molecules,constitutes a potential clue for new therapeutical approaches to thesediseases. In view of the suspected central role of MACHα1 in FAS-R andTNF toxicity, it seems particularly important to design drugs that canblock the proteolytic function of this molecule, as has been done forsome other members of the CED3/ICE family. The unique sequence featuresof the CED3/ICE homolog encompassed in the MACHα1 molecules may allowdesigning drugs that can affect its protection from excessiveimmune-mediated cytotoxicity without interfering with physiological celldeath processes, in which other members of the CED3/ICE family areinvolved.

Thus, the present invention also concerns the DNA sequence encoding aMORT-1-binding protein and the MORT-1-binding proteins encoded by theDNA sequences.

Moreover, the present invention further concerns the DNA sequencesencoding biologically active analogs, fragments and derivatives of theMORT-1-binding protein, and the analogs, fragments and derivativesencoded thereby. The preparation of such analogs, fragments andderivatives is by standard procedure (see for example, Sambrook et al.,1989) in which in the DNA sequences encoding the MORT-1-binding protein,one or more codons may be deleted, added or substituted by another, toyield analogs having at least one amino acid residue change with respectto the native protein.

A polypeptide or protein “substantially corresponding” to MORT-1-bindingprotein includes not only MORT-1-binding protein but also polypeptidesor proteins that are analogs of MORT-1-binding.

Analogs that substantially correspond to MORT-1-binding protein arethose polypeptides in which one or more amino acid of the MORT-1-bindingprotein's amino acid sequence has been replaced with another amino acid,deleted and/or inserted, provided that the resulting protein exhibitssubstantially the same or higher biological activity as theMORT-1-binding protein to which it corresponds.

In order to substantially correspond to MORT-1-binding protein, thechanges in the sequence of MORT-1-binding proteins, such as MACHisoforms are generally relatively minor. Although the number of changesmay be more than ten, preferably there are no more than ten changes,more preferably no more than five, and most preferably no more thanthree such changes. While any technique can be used to find potentiallybiologically active proteins which substantially correspond toMORT-1-binding proteins, one such technique is the use of conventionalmutagenesis techniques on the DNA encoding the protein, resulting in afew modifications. The proteins expressed by such clones can then bescreened for MORT-1 binding and/or FAS-R and p55-R mediating activity.

“Conservative” changes are those changes which would not be expected tochange the activity of the protein and are usually the first to bescreened as these would not be expected to substantially change thesize, charge or configuration of the protein and thus would not beexpected to change the biological properties thereof.

Conservative substitutions of MORT-1-binding proteins include an analogwherein at least one amino acid residue in the polypeptide has beenconservatively replaced by a different amino acid. Such substitutionspreferably are made in accordance with the following list as presentedin Table IA, which substitutions may be determined by routineexperimentation to provide modified structural and functional propertiesof a synthesized polypeptide molecule while maintaining the biologicalactivity characteristic of MORT-1-binding protein.

TABLE IA Original Exemplary Residue Substitution Ala Gly; Ser Arg LysAsn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala; Pro His Asn; GlnIle Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Tyr; Ile Phe Met;Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Alternatively, another group of substitutions of MORT-1-binding proteinare those in which at least one amino acid residue in the polypeptidehas been removed and a different residue inserted in its place accordingto the following Table IB. The types of substitutions which may be madein the polypeptide may be based on analysis of the frequencies of aminoacid changes between a homologous protein of different species, such asthose presented in Table 1-2 of Schulz et al., G. E., Principles ofProtein Structure Springer-Verlag, New York, N.Y., 1798, and FIGS. 3-9of Creighton, T. E., Proteins: Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, Calif. 1983. Based on such an analysis,alternative conservative substitutions are defined herein as exchangeswithin one of the following five groups:

TABLE IB 1. Small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr (Pro, Gly); 2. Polar negatively charged residues and theiramides: Asp, Asn, Glu, Gln; 3. Polar, positively charged residues: His,Arg, Lys; 4. Large aliphatic nonpolar residues: Met, Leu, Ile, Val(Cys); and 5. Large aromatic residues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking any side chain andthus imparts flexibility to the chain. This however tends to promote theformation of secondary structure other than α-helical. Pro, because ofits unusual geometry, tightly constrains the chain and generally tendsto promote β-turn-like structures, although in some cases Cys can becapable of participating in disulfide bond formation which is importantin protein folding. Note that Schulz et al., supra, would merge Groups 1and 2, above. Note also that Tyr, because of its hydrogen bondingpotential, has significant kinship with Ser, and Thr, etc.

Conservative amino acid substitutions according to the presentinvention, e.g., as presented above, are known in the art and would beexpected to maintain biological and structural properties of thepolypeptide after amino acid substitution. Most deletions andsubstitutions according to the present invention are those which do notproduce radical changes in the characteristics of the protein orpolypeptide molecule. “Characteristics” is defined in a non-inclusivemanner to define both changes in secondary structure, e.g. α-helix orβ-sheet, as well as changes in biological activity, e.g., binding ofMORT-1 or mediation of FAS-R ligand or TNF effect on cells.

Examples of production of amino acid substitutions in proteins which canbe used for obtaining analogs of MORT-1-binding proteins for use in thepresent invention include any known method steps, such as presented inU.S. Pat. Nos. RE 33,653, 4,959,314, 4,588,585 and U.S. Pat. No.4,737,462, to Mark et al.; U.S. Pat. No. 5,116,943 to Koths et al., U.S.Pat. No. 4,965,195 to Namen et al.; U.S. Pat. No. 4,879,111 to Chong etal.; and U.S. Pat. No. 5,017,691 to Lee et al.; and lysine substitutedproteins presented in U.S. Pat. No. 4,904,584 (Shaw et al.).

Besides conservative substitutions discussed above which would notsignificantly change the activity of MORT-1-binding protein, eitherconservative substitutions or less conservative and more random changes,which lead to an increase in biological activity of the analogs ofMORT-1-binding proteins, are intended to be within the scope of theinvention.

When the exact effect of the substitution or deletion is to beconfirmed, one skilled in the art will appreciate that the effect of thesubstitution(s), deletion(s), etc., will be evaluated by routine bindingand cell death assays. Screening using such a standard test does notinvolve undue experimentation.

Acceptable analogs are those which retain at least the capability ofbinding to MORT-1, and thereby, as noted above mediate the activity(e.g., by the protease activity of at least some of the MACH isoforms)of the FAS-R and p55-R. In such a way, analogs can be produced whichhave a so-called dominant-negative effect, namely, an analog which isdefective either in binding to MORT-1, or in subsequent signaling orprotease activity following such binding. Such analogs can be used, forexample, to inhibit the FAS-ligand-effect by competing with the naturalMORT-1-binding proteins. For example, it appears likely that the MACHisoforms, MACHα2 and MACHα3 are “natural” analogs which serve to inhibitMACH activity by competing with the binding of the active (protease)MACH isoforms to MORT-1 which appears to be essential for the activationof these MACH isoforms. Once the active MACH isoforms cannot bind toMORT-1, the intracellular signaling pathways mediated by FAS-R and p55-Rwill thereby also be inhibited. Likewise, so-called dominant-positiveanalogs may be produced which would serve to enhance the FAS ligand orTNF effect. These would have the same or better MORT-1-bindingproperties and the same or better signaling properties of the naturalMORT-1-binding proteins.

At the genetic level, these analogs are generally prepared bysite-directed mutagenesis of nucleotides in the DNA encoding theMORT-1-binding protein, thereby producing DNA encoding the analog, andthereafter synthesizing the DNA and expressing the polypeptide inrecombinant cell culture. The analogs typically exhibit the same orincreased qualitative biological activity as the naturally occurringprotein, Ausubel et al., Current Protocols in Molecular Biology, GreenePublications and Wiley Interscience, New York, N.Y., 1987-1995; Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989.

Preparation of a MORT-1-binding protein in accordance herewith, or analternative nucleotide sequence encoding the same polypeptide butdiffering from the natural sequence due to changes permitted by theknown degeneracy of the genetic code, can be achieved by site-specificmutagenesis of DNA that encodes an earlier prepared analog or a nativeversion of a MORT-1-binding protein. Site-specific mutagenesis allowsthe production of analogs through the use of specific oligonucleotidesequences that encode the DNA sequence of the desired mutation, as wellas a sufficient number of adjacent nucleotides, to provide a primersequence of sufficient size and sequence complexity to form a stableduplex on both sides of the deletion junction being traversed.Typically, a primer of about 20 to 25 nucleotides in length ispreferred, with about 5 to 10 complementing nucleotides on each side ofthe sequence being altered. In general, the technique of site-specificmutagenesis is well known in the art, as exemplified by publicationssuch as Adelman et al., DNA 2:183 (1983), the disclosure of which isincorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily available commercially and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153:3, 1987) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevantpolypeptide. An oligonucleotide primer bearing the desired mutatedsequence is prepared synthetically by automated DNA/oligonucleotidesynthesis. This primer is then annealed with the single-strandedprotein-sequence-containing vector, and subjected to DNA-polymerizingenzymes such as E. coli polymerase I Klenow fragment, to complete thesynthesis of the mutation-bearing strand. Thus, a mutated sequence andthe second strand bears the desired mutation. This heteroduplex vectoris then used to transform appropriate cells, such as E. coli JM101cells, and clones are selected that include recombinant vectors bearingthe mutated sequence arrangement.

After such a clone is selected, the mutated MORT-1-binding protein maybe removed and placed in an appropriate vector, generally a transfer orexpression vector of the type that may be employed for transfection ofan appropriate host.

Accordingly, gene or nucleic acid encoding for a MORT-1-binding proteincan also be detected, obtained and/or modified, in vitro, in situ and/orin vivo, by the use of known DNA or RNA amplification techniques, suchas PCR and chemical oligonucleotide synthesis. PCR allows for theamplification (increase in number) of specific DNA sequences by repeatedDNA polymerase reactions. This reaction can be used as a replacement forcloning; all that is required is a knowledge of the nucleic acidsequence. In order to carry out PCR, primers are designed which arecomplementary to the sequence of interest. The primers are thengenerated by automated DNA synthesis. Because primers can be designed tohybridize to any part of the gene, conditions can be created such thatmismatches in complementary base pairing can be tolerated. Amplificationof these mismatched regions can lead to the synthesis of a mutagenizedproduct resulting in the generation of a peptide with new properties(i.e., site directed mutagenesis). See also, e.g., Ausubel, supra, Ch.16. Also, by coupling complementary DNA (cDNA) synthesis, using reversetranscriptase, with PCR, RNA can be used as the starting material forthe synthesis of the extracellular domain of a prolactin receptorwithout cloning.

Furthermore, PCR primers can be designed to incorporate new restrictionsites or other features such as termination codons at the ends of thegene segment to be amplified. This placement of restriction sites at the5′ and 3′ ends of the amplified gene sequence allows for gene segmentsencoding MORT-1-binding protein or a fragment thereof to be customdesigned for ligation other sequences and/or cloning sites in vectors.

PCR and other methods of amplification of RNA and/or DNA are well knownin the art and can be used according to the present invention withoutundue experimentation, based on the teaching and guidance presentedherein. Known methods of DNA or RNA amplification include, but are notlimited to polymerase chain reaction (PCR) and related amplificationprocesses (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159,4,965,188, to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 toTabor et al.; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464to Wilson et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No.5,066,584 to Gyllensten et al.; U.S. Pat. No. 4,889,818 to Gelfand etal.; U.S. Pat No. 4,994,370 to Silver et al.; U.S. Pat. No. 4,766,067 toBiswas; U.S. Pat. No. 4,656,134 to Ringold; and Innis et al., eds., PCRProtocols: A Guide to Method and Applications) and RNA mediatedamplification which uses anti-sense RNA to the target sequence as atemplate for double stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek et al., with the tradename NASBA); and immuno-PCR which combinesthe use of DNA amplification with antibody labeling (Ruzicka et al.,Science 260:487 (1993); Sano et al., Science 258:120 (1992); Sano etal., Biotechniques 9:1378 (1991)), the entire contents of which patentsand reference are entirely incorporated herein by reference.

In an analogous fashion, biologically active fragments of MORT-1-bindingproteins (e.g., those of any of the MACH isoforms) may be prepared asnoted above with respect to the analogs of MORT-1-binding proteins.Suitable fragments of MORT-1-binding proteins are those which retain theMORT-1 binding capability and which can mediate the biological activityof FAS-R and p55-R as noted above. Accordingly, MORT-1-binding proteinfragments can be prepared which have a dominant-negative or adominant-positive effect as noted above with respect to the analogs. Itshould be noted that these fragments represent a special class of theanalogs of the invention, namely, they are defined portions ofMORT-1-binding proteins derived from the full MORT-1-binding proteinsequence (e.g., from that of any one of the MACH isoforms), each suchportion or fragment having any of the above-noted desired activities.Such fragment may be, e.g., a peptide.

Similarly, derivatives may be prepared by standard modifications of theside groups of one or more amino acid residues of the MORT-1-bindingprotein, its analogs or fragments, or by conjugation of theMORT-1-binding protein, its analogs or fragments, to another moleculee.g. an antibody, enzyme, receptor, etc., as are well known in the art.Accordingly, “derivatives” as used herein covers derivatives which maybe prepared from the functional groups which occur as side chains on theresidues or the N- or C-terminal groups, by means known in the art, andare included in the invention. Derivatives may have chemical moietiessuch as carbohydrate or phosphate residues, provided such a fraction hasthe same or higher biological activity as MORT-1-binding proteins.

For example, derivatives may include aliphatic esters of the carboxylgroups, amides of the carboxyl groups by reaction with ammonia or withprimary or secondary amines, N-acyl derivatives or free amino groups ofthe amino acid residues formed with acyl moieties (e.g., alkanoyl orcarbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group(for example that of seryl or threonyl residues) formed with acylmoieties.

The term “derivatives” is intended to include only those derivativesthat do not change one amino acid to another of the twenty commonlyoccurring natural amino acids.

Although MORT-1-binding protein is a protein or polypeptide, it is asequence of amino acid residues. A polypeptide consisting of a largersequence which includes the entire sequence of a MORT-1-binding protein,in accordance with the definitions herein, is intended to be includedwithin the scope of such a polypeptide as long as the additions do notaffect the basic and novel characteristics of the invention, i.e., ifthey either retain or increase the biological activity of MORT-1-bindingprotein or can be cleaved to leave a protein or polypeptide having thebiological activity of MORT-1-binding protein. Thus, for example, thepresent invention is intended to include fusion proteins ofMORT-1-binding protein with other amino acids or peptides.

The new MORT-1-binding protein, their analogs, fragments and derivativesthereof, have a number of uses, for example:

(i) MORT-1-binding protein, its analogs, fragments and derivativesthereof, may be used to mimic or enhance the function of MORT-1 andhence the FAS-R ligand or TNF, in situations where an enhanced FAS-Rligand or TNF effect is desired, such as in anti-tumor,anti-inflammatory, anti-HIV applications, etc., where the FAS-R ligand-or TNF-induced cytotoxicity is desired. In this case the MORT-1-bindingprotein, its analogs, fragments or derivatives thereof, which enhancethe FAS-R ligand of TNF effect, i.e., cytotoxic effect, may beintroduced to the cells by standard procedures known per se. Forexample, as the MORT-1-binding protein are intracellular and should beintroduced only into the cells where the FAS-R ligand or TNF effect isdesired, a system for specific introduction of this protein into thecells is necessary. One way of doing this is by creating a recombinantanimal virus, e.g., one derived from Vaccinia, to the DNA of which thefollowing two genes will be introduced: the gene encoding a ligand thatbinds to cell surface proteins specifically expressed by the cells,e.g., ones such as the AIDs (HIV) virus gp120 protein which bindsspecifically to some cells (CD4 lymphocytes and related leukemias), orany other ligand that binds specifically to cells carrying a FAS-R orp55-R, such that the recombinant virus vector will be capable of bindingsuch FAS-R- or p55-R-carrying cells; and the gene encoding theMORT-1-binding protein. Thus, expression of the cell-surface-bindingprotein on the surface of the virus will target the virus specificallyto the tumor cell or other FAS-R- or p55-R-carrying cell, followingwhich the MORT-1-binding protein encoding sequence will be introducedinto the cells via the virus, and once expressed in the cells, willresult in enhancement of the FAS-R ligand or TNF effect leading to thedeath of the tumor cells or other FAS-R- or p55-R-carrying cells it isdesired to kill. Construction of such recombinant animal virus is bystandard procedures (see for example, Sambrook et al., 1989). Anotherpossibility is to introduce the sequences of the MORT-1-binding protein(e.g., any one of the MACH isoforms) in the form of oligonucleotideswhich can be absorbed by the cells and expressed therein.

(ii) They may be used to inhibit the FAS-R ligand or TNF effect, e.g.,in cases such as tissue damage in septic shock, graft-vs.-hostrejection, or acute hepatitis, in which it is desired to block the FAS-Rligand or TNF induced FAS-R or p55-R intracellular signaling. In thissituation, it is possible, for example, to introduce into the cells, bystandard procedures, oligonucleotides having the anti-sense codingsequence for the MORT-1-binding protein, which would effectively blockthe translation of mRNAs encoding either the MORT-1 protein or theMORT-1-binding protein and thereby block its expression and lead to theinhibition of the FAS-R ligand- or TNF-effect. Such oligonucleotides maybe introduced into the cells using the above recombinant virus approach,the second sequence carried by the virus being the oligonucleotidesequence.

Another possibility is to use antibodies specific for the MORT-1-bindingprotein to inhibit its intracellular signaling activity.

Yet another way of inhibiting the FAS-R ligand or TNF effect is by therecently developed ribozyme approach. Ribozymes are catalytic RNAmolecules that specifically cleave RNAs. Ribozymes may be engineered tocleave target RNAs of choice, e.g., the mRNAs encoding theMORT-1-binding protein of the invention. Such ribozymes would have asequence specific for the MORT-1-binding protein mRNA and would becapable of interacting therewith (complementary binding) followed bycleavage of the mRNA, resulting in a decrease (or complete loss) in theexpression of the MORT-1-binding protein, the level of decreasedexpression being dependent upon the level of ribozyme expression in thetarget cell. To introduce ribozymes into the cells of choice (e.g.,those carrying FAS-R or p55-R), any suitable vector may be used, e.g.,plasmid, animal virus (retrovirus) vectors, that are usually used forthis purpose (see also (i) above, where the virus has, as secondsequence, a cDNA encoding the ribozyme sequence of choice). (Forreviews, methods etc. concerning ribozymes see Chen et al., 1992; Zhaoand Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama etal., 1993; Cantor et al., 1993; Barinaga, 1993; Crisell et al., 1993 andKoizumi et al., 1993).

(iii) The MORT-1-binding protein, its analogs, fragments or derivativesmay also be used to isolate, identify and clone other proteins of thesame class, i.e., those binding to FAS-R intracellular domain or tofunctionally related receptors, or those binding to MORT-1 and therebyto functionally related receptors such as FAS-R and p55-R, and involvedin the intracellular signaling process. In this application the abovenoted yeast two-hybrid system may be used, or there may be used arecently developed system employing non-stringent Southern hybridizationfollowed by PCR cloning (Wilks et al., 1989). In the Wilks et al.publication, there is described the identification and cloning of twoputative protein-tyrosine kinases by application of non-stringentsouthern hybridization followed by cloning by PCR based on the knownsequence of the kinase motif, a conceived kinase sequence. This approachmay be used, in accordance with the present invention using the sequenceof the MORT-1-binding protein (e.g., any of the MACH isoforms) toidentify and clone those of related MORT-1-binding proteins.

(iv) Yet another approach to utilizing the MORT-1-binding protein, orits analogs, fragments or derivatives thereof, of the invention is touse them in methods of affinity chromatography to isolate and identifyother proteins or factors to which they are capable of binding, e.g.,MORT-1, or other proteins or factors involved in the intracellularsignaling process. In this application, the MORT-1-binding protein, itsanalogs, fragments or derivatives thereof, of the present invention, maybe individually attached to affinity chromatography matrices and thenbrought into contact with cell extracts or isolated proteins or factorssuspected of being involved in the intracellular signaling process.Following the affinity chromatography procedure, the other proteins orfactors which bind to the MORT-1-binding protein, or its analogs,fragments or derivatives thereof of the invention, can be eluted,isolated and characterized.

(v) As noted above, the MORT-1-binding protein, or its analogs,fragments or derivatives thereof, of the invention may also be used asimmunogens (antigens) to produce specific antibodies thereto. Theseantibodies may also be used for the purposes of purification of theMORT-1-binding protein (e.g., MACH isoforms) either from cell extractsor from transformed cell lines producing MORT-1-binding protein, or itsanalogs or fragments. Further, these antibodies may be used fordiagnostic purposes for identifying disorders related to abnormalfunctioning of the FAS-R ligand or TNF system, e.g., overactive orunderactive FAS-R ligand- or TNF-induced cellular effects. Thus, shouldsuch disorders be related to a malfunctioning intracellular signalingsystem involving the MORT-1 protein, or MORT-1-binding protein, suchantibodies would serve as an important diagnostic tool.

It should also be noted that the isolation, identification andcharacterization of the MORT-1-binding protein (e.g., the MACH isoforms)of the invention may be performed using any of the well known standardscreening procedures. For example, one of these screening procedures,the yeast two-hybrid procedure as is set forth herein (Example 1), wasused to identify the MORT-1 protein and subsequently the MORT-1-bindingproteins (Examples 2-3) of the invention. Likewise as noted above andbelow, other procedures may be employed such as affinity chromatography,DNA hybridization procedures, etc. as are well known in the art, toisolate, identify and characterize MORT-1-binding protein of theinvention or to isolate, identify and characterize additional proteins,factors, receptors, etc. which are capable of binding to the MORT-1protein or to the MORT-1-binding proteins of the invention.

As set forth hereinabove, the MORT-1-binding protein may be used togenerate antibodies specific to MORT-1-binding proteins, e.g., MACHisoforms. These antibodies or fragments thereof may be used as set forthhereinbelow in detail, it being understood that in these applicationsthe antibodies or fragments thereof are those specific forMORT-1-binding proteins.

Based on the findings in accordance with the present invention that atleast some of the MACH isoforms (see above and Example 3 below) areproteases related to the proteases of the CED3/ICE family of proteases,the following specific medical applications are envisioned for theseMACH isoforms: it has been found that specific inhibitors of otherCED3/ICE proteases, some of which are cell permeable, already exist,which can block effectively programmed cell death processes. Hence, itis possible in accordance with the present invention to designinhibitors that can prevent FAS-R ligand- or TNF-induced cell death, thepathways in which the MACH protease isoforms are involved. Further, inview of the unique sequence features of these new MACH proteases, itseems possible to design inhibitors that will be highly specific to theTNF- and FAS-R ligand-induced effects. The findings of the presentinvention also provide a way to study the mechanism in which the“killing protease” is activated in response to FAS-R ligand and TNF,this allowing subsequent development of drugs that can control theextent of this activation. There are many diseases in which such drugscan be of great help. Amongst others, acute hepatitis in which the acutedamage to the liver seems to reflect FAS-R ligand-mediated death of theliver cells; autoimmune-induced cell death such as the death of the βLangerhans cells of the pancreas, that results in diabetes; the death ofcells in graft rejection (e.g., kidney, heart and liver); the death ofoligodendrocytes in the brain in multiple sclerosis; and AIDS-inhibitedT cell suicide which causes proliferation of the AIDS virus and hencethe AIDS disease.

As mentioned hereinabove and hereinbelow, it appears that two of theMACH isoforms, MACHα2 and MACHα3 may serve as “natural” inhibitors ofthe MACH protease isoforms, and these may thus be employed as the abovenoted specific inhibitors of these MACH proteases. Likewise, othersubstances such as peptides, organic compounds, antibodies, etc. mayalso be screened to obtain specific drugs which are capable ofinhibiting the MACH proteases.

A non-limiting example of how peptide inhibitors of the MACH proteaseswould be designed and screened is based on previous studies on peptideinhibitors of ICE or ICE-like proteases, the substrate specificity ofICE and strategies for epitope analysis using peptide synthesis. Theminimum requirement for efficient cleavage of peptide by ICE was foundto involve four amino acids to the left of the cleavage site with astrong preference for aspartic acid in the P₁ position and withmethylamine being sufficient to the right of the P₁ position (Sleath etal., 1990; Howard et al., 1991; Thornberry et al., 1992). Furthermore,the fluorogenic substrate peptide (a tetrapeptide),acetyl-Asp-Glu-Val-Asp-a-(4-methyl-coumaryl-7-amide) abbreviatedAc-DEVD-AMC, corresponds to a sequence in poly (ADP-ribose) polymerase(PARP) found to be cleaved in cells shortly after FAS-R stimulation, aswell as other apoptopic processes (Kaufmann, 1989; Kaufmann et al.,1993; Lazebnik et al., 1994), and is cleaved effectively by CPP32 (amember of the CED3/ICE protease family) and MACH proteases.

As Asp in the P₁ position of the substrate appears to be important,tetrapeptides having Asp as the fourth amino acid residue and variouscombinations of amino acids in the first three residue positions can berapidly screened for binding to the active site of MACH proteases using,for example, the method developed by Geysen (Geysen, 1985; Geysen etal., 1987) where a large number of peptides on solid supports werescreened for specific interactions with antibodies. The binding of MACHproteases to specific peptides can be detected by a variety of wellknown detection methods within the skill of those in the art, such asradiolabeling of the MACH protease, etc. This method of Geysen's wasshown to be capable of testing at least 4000 peptides each working day.

Since it may be advantageous to design peptide inhibitors thatselectively inhibit MACH proteases without interfering withphysiological cell death processes in which other members of theCED3/ICE family of proteases are involved, the pool of peptides bindingto MACH proteases in an assay such as the one described above can befurther synthesized as a fluorogenic substrate peptide to test forselective cleavage by MACH proteases without being cleaved by otherCED3/ICE proteases. Peptides which are determined to be selectivelycleaved by the MACH proteases, can then be modified to enhance cellpermeability and inhibit the cell death activity of MACH eitherreversibly or irreversibly. Thornberry et al. (1994) reported that atetrapeptide (acyloxy) methyl ketoneAc-Tyr-Val-Ala-Asp-CH₂OC(O)-[2,6-(CF₃)₂]Ph was a potent inactivator ofICE. Similarly, Milligan et al. (1995) reported that tetrapeptideinhibitors having a chloromethylketone (irreversibly) or aldehyde(reversibly) groups inhibited ICE. In addition, abenzyloxycarboxyl-Asp-CH₂OC(O)-2,6-dichlorobenzene (DCB) was shown toinhibit ICE (Mashima et al., 1995). Accordingly, tetrapeptides thatselectively bind to MACH proteases can be modified with, for example, analdehyde group, chloromethylketone, (acyloxy) methyl ketone or aCH₂OC(0)-DCB group to create a peptide inhibitor of MACH proteaseactivity.

While some specific inhibitors of other CED3/ICE proteases are cellpermeable, the cell permeability of peptide inhibitors may need to beenhanced. For instance, peptides can be chemically modified orderivatized to enhance their permeability across the cell membrane andfacilitate the transport of such peptides through the membrane and intothe cytoplasm. Muranishi et al. (1991) reported derivatizingthyrotropin-releasing hormone with lauric acid to form a lipophiliclauroyl derivative with good penetration characteristics across cellmembranes. Zacharia et al. (1991) also reported the oxidation ofmethionine to sulfoxide and the replacement of the peptide bond with itsketomethylene isoester (COCH₂) to facilitate transport of peptidesthrough the cell membrane. These are just some of the knownmodifications and derivatives that are well within the skill of those inthe art.

Furthermore, drug or peptide inhibitors, which are capable of inhibitingthe cell death activity of MACHα1 and MACHα2, can be conjugated orcomplexed with molecules that facilitate entry into the cell.

U.S. Pat. No. 5,149,782 discloses conjugating a molecule to betransported across the cell membrane with a membrane blending agent suchas fusogenic polypeptides, ion-channel forming polypeptides, othermembrane polypeptides, and long chain fatty acids, e.g., myristic acid,palmitic acid. These membranes blending agents insert the molecularconjugates into the lipid bilayer of cellular membranes and facilitatetheir entry into the cytoplasm.

Low et al., U.S. Pat. No. 5,108,921, reviews available methods fortransmembrane delivery of molecules such as, but not limited to,proteins and nucleic acids by the mechanism of receptor mediatedendocytotic activity. These receptor systems include those recognizinggalactose, mannose, mannose 6-phosphate, transferrin,asialoglycoprotein, transcobalamin (vitamin B₁₂), α-2 macroglobulins,insulin and other peptide growth factors such as epidermal growth factor(EGF). Low et al. teaches that nutrient receptors, such as receptors forbiotin and folate, can be advantageously used to enhance transportacross the cell membrane due to the location and multiplicity of biotinand folate receptors on the membrane surfaces of most cells and theassociated receptor mediated transmembrane transport processes. Thus, acomplex formed between a compound to be delivered into the cytoplasm anda ligand, such as biotin or folate, is contacted with a cell membranebearing biotin or folate receptors to initiate the receptor mediatedtrans-membrane transport mechanism and thereby permit entry of thedesired compound into the cell.

ICE is known to have the ability to tolerate liberal substitutions inthe P₂ position and this tolerance to liberal substitutions wasexploited to develop a potent and highly selective affinity labelcontaining a biotin tag (Thornberry et al., 1994). Consequently, the P₂position as well as possibly the N-terminus of the tetrapeptideinhibitor can be modified or derivatized, such as to with the additionof a biotin molecule, to enhance the permeability of these peptideinhibitors across the cell membrane.

In addition, it is known in the art that fusing a desired peptidesequence with a leader/signal peptide sequence to create a “chimericpeptide” will enable such a “chimeric peptide” to be transported acrossthe cell membrane into the cytoplasm.

As will be appreciated by those of skill in the art of peptides, thepeptide inhibitors of MACH proteolytic activity according to the presentinvention is meant to include peptidomimetic drugs or inhibitors, whichcan also be rapidly screened for binding to MACH protease to designperhaps more stable inhibitors.

It will also be appreciated that the same means for facilitating orenhancing the transport of peptide inhibitors across cell membranes asdiscussed above are also applicable to the MACH isoforms themselves aswell as other peptides and proteins which exerts their effectsintracellularly.

As regards the antibodies mentioned herein throughout, the term“antibody” is meant to include polyclonal antibodies, monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id)antibodies to antibodies that can be labeled in soluble or bound form,as well as fragments thereof provided by any known technique, such as,but not limited to enzymatic cleavage, peptide synthesis or recombinanttechniques.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which populations containssubstantially similar epitope binding sites. MAbs may be obtained bymethods known to those skilled in the art. See, for example Kohler andMilstein, Nature, 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubelet al., eds., Harlow and Lane ANTIBODIES A LABORATORY MANUAL, ColdSpring Harbor Laboratory (1988); and Colligan et al., eds., CurrentProtocols in Immunology, Greene Publishing Assoc. and Wiley InterscienceN.Y., (1992-1996), the contents of which references are incorporatedentirely herein by reference. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclassthereof. A hybridoma producing a mAb of the present invention may becultivated in vitro, in situ or in vivo. Production of high titers ofmAbs in vivo or in situ makes this the presently preferred method ofproduction.

Chimeric antibodies are molecules of which different portions arederived from different animal species, such as those having the variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine mabs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mabs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277(1984);′Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984);Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., EuropeanPatent Application 125023 (published Nov. 14, 1984); Neuberger et al.,Nature 314:268-270 (1985); Taniguchi et al., European Patent Application171496 (published Feb. 19, 1985); Morrison et al., European PatentApplication 173494 (published Mar. 5, 1986); Neuberger et al., PCTApplication WO 8601533, (published Mar. 13, 1986); Kudo et al., EuropeanPatent Application 184187 (published Jun. 11, 1986); Sahagan et al., J.Immunol. 137:1066-1074 (1986); Robinson et al., International PatentApplication No. WO8702671 (published May 7, 1987); Liu et al., Proc.Natl. Acad. Sci USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad.Sci USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988);and Harlow and Lane, ANTIBODIES:A LABORATORY MANUAL, supra. Thesereferences are entirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g. mouse strain) as the sourceof the mAb to which an anti-Id is being prepared. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody to these idiotypicdeterminants (the anti-Id antibody). See, for example, U.S. Pat. No.4,699,880, which is herein entirely incorporated by reference.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the MORT-1-binding proteins,analogs, fragments or derivatives thereof, of the present invention maybe used to induce anti-Id antibodies in suitable animals, such as BALB/cmice. Spleen cells from such immunized mice are used to produce anti-Idhybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can becoupled to a carrier such as keyhole limpet hemocyanin (KLH) and used toimmunize additional BALB/c mice. Sera from these mice will containanti-anti-Id antibodies that have the binding properties of the originalmAb specific for an epitope of the above MORT-1-binding protein, oranalogs, fragments and derivatives thereof.

The anti-Id mAbs thus have their own idiotypic epitopes, or “idiotopes”structurally similar to the epitope being evaluated, such as GRBprotein-a.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab′)2, whichare capable of binding antigen. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies useful in the present invention may be used for the detectionand quantitation of the MORT-1-binding protein according to the methodsdisclosed herein for intact antibody molecules. Such fragments aretypically produced by proteolytic cleavage, using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The antibodies, including fragments of antibodies, useful in the presentinvention may be used to quantitatively or qualitatively detect theMORT-1-binding protein in a sample or to detect presence of cells whichexpress the MORT-1-binding protein of the present invention. This can beaccomplished by immunofluorescence techniques employing a fluorescentlylabeled antibody (see below) coupled with light microscopic, flowcytometric, or fluorometric detection.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of the MORT-1-bindingprotein of the present invention. In situ detection may be accomplishedby removing a histological specimen from a patient, and providing thelabeled antibody of the present invention to such a specimen. Theantibody (or fragment) is preferably provided by applying or byoverlaying the labeled antibody (or fragment) to a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of the MORT-1-binding protein, but also itsdistribution on the examined tissue. Using the present invention, thoseof ordinary skill will readily perceive that any of wide variety ofhistological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Such assays for the MORT-1-binding protein of the present inventiontypically comprises incubating a biological sample, such as a biologicalfluid, a tissue extract, freshly harvested cells such as lymphocytes orleukocytes, or cells which have been incubated in tissue culture, in thepresence of a detectably labeled antibody capable of identifying theMORT-1-binding protein, and detecting the antibody by any of a number oftechniques well known in the art.

The biological sample may be treated with a solid phase support orcarrier such as nitrocellulose, or other solid support or carrier whichis capable of immobilizing cells, cell particles or soluble proteins.The support or carrier may then be washed with suitable buffers followedby treatment with a detectably labeled antibody in accordance with thepresent invention, as noted above. The solid phase support or carriermay then be washed with the buffer a second time to remove unboundantibody. The amount of bound label on said solid support or carrier maythen be detected by conventional means.

By “solid phase support”, “solid phase carrier”, “solid support”, “solidcarrier”, “support” or “carrier” is intended any support or carriercapable of binding antigen or antibodies. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros and magnetite. The nature of the carrier can beeither soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know may other suitable carriers for binding antibody orantigen, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of antibody, of the invention asnoted above, may be determined according to well known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which an antibody in accordance with the presentinvention can be detectably labeled is by linking the same to an enzymeand used in an enzyme immunoassay (EIA). This enzyme, in turn, whenlater exposed to an appropriate substrate, will react with the substratein such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorometric or by visual means.Enzymes which can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomeras, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholin-esterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactive labeling the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work,T.S. et al., North Holland Publishing Company, NY (1978) with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, T., incorporated by reference herein.The radioactive isotope can be detected by such means as the use of a gcounter or a scintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the presentinvention with a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canbe then detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²E, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the antigen from the sample by formationof a binary solid phase antibody-antigen complex. After a suitableincubation period, the solid support or carrier is washed to remove theresidue of the fluid sample, including unreacted antigen, if any, andthen contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support or carrier through the unlabeledantibody, the solid support or carrier is washed a second time to removethe unreacted labeled antibody.

In another type of “sandwich” assay, which may also be useful with theantigens of the present invention, the so-called “simultaneous” and“reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support or carrierand labeled antibody are both added to the sample being tested at thesame time. After the incubation is completed, the solid support orcarrier is washed to remove the residue of fluid sample and uncomplexedlabeled antibody. The presence of labeled antibody associated with thesolid support or carrier is then determined as it would be in aconventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support or carrier after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support or carrier is thendetermined as in the “simultaneous” and “forward” assays.

The MORT-1-binding proteins of the invention may be produced by anystandard recombinant DNA procedure (see for example, Sambrook, et al.,1989 and Ansabel et al., 1987-1995, supra) in which suitable eukaryoticor prokaryotic host cells well known in the art are transformed byappropriate eukaryotic or prokaryotic vectors containing the sequencesencoding for the proteins. Accordingly, the present invention alsoconcerns such expression vectors and transformed hosts for theproduction of the proteins of the invention. As mentioned above, theseproteins also include their biologically active analogs, fragments andderivatives, and thus the vectors encoding them also include vectorsencoding analogs and fragments of these proteins, and the transformedhosts include those producing such analogs and fragments. Thederivatives of these proteins, produced by the transformed hosts, arethe derivatives produced by standard modification of the proteins ortheir analogs or fragments.

The present invention also relates to pharmaceutical compositionscomprising recombinant animal virus vectors encoding the MORT-1-bindingproteins, which vector also encodes a virus surface protein capable ofbinding specific target cell (e.g., cancer cells) surface proteins todirect the insertion of the MORT-1-binding protein sequences into thecells. Further pharmaceutical compositions of the invention comprises asthe active ingredient (a) an oligonucleotide sequence encoding ananti-sense sequence of the MORT-1-binding protein sequence, or (b) drugsthat block the proteolytic activity of MACH isoforms.

Pharmaceutical compositions according to the present invention include asufficient amount of the active ingredient to achieve its intendedpurpose. In addition, the pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically and which can stabilizesuch preparations for administration to the subject in need thereof aswell known to those of skill in the art.

The MORT-1 binding protein MACH, is expressed in different tissues atmarkedly different levels and apparently also with different patterns ofisotypes. These differences probably contribute to the tissue-specificfeatures of response to the Fas/APO1-ligand and TNF. As in the case ofother CED3/ICE homologs (Wang et al., 1994; Alnemri et al., 1995), MACHisoforms that contain incomplete CED3/ICE regions (e.g., MACHα3) arefound to have an inhibitory effect on the activity of co-expressedMACHα1 or MACHα2 molecules; they are also found to block death inductionby Fas/APO1 and p55-R. Expression of such inhibitory isoforms in cellsmay constitute a mechanism of cellular self-protection against Fas/APO1-and TNF-mediated cytotoxicity. The wide heterogeneity of MACH isoforms,which greatly exceeds that observed for any of the other proteases ofthe CED3/ICE family, should allow a particularly fine tuning of thefunction of the active MACH isoforms.

It is also possible that some of the MACH isoforms serve otherfunctions. The ability of MACHβ1 to bind to both MORT1 and MACHα1suggests that this isoform could actually enhance the activity of theenzymatically active isoforms. The mild cytotoxicity observed in293-EBNA and MCF7 cultures transfected with this isoform and the rathersignificant cytotoxic effect that it exerts in HeLa cells are likely toreflect activation of endogenously-expressed MACHA molecules uponbinding to the transfected MACHβ1 molecules. Conceivably, some of theMACH isoforms could also act as docking sites for molecules that areinvolved in other, non-cytotoxic effects of Fas/APO1 and TNF receptors.

Due to the unique ability of Fas/APO1 and TNF receptors to cause celldeath, as well as the ability of the TNF receptors to trigger othertissue-damaging activities, aberrations in the function of thesereceptors could be particularly deleterious to the organism. Indeed,both excessive and deficient functioning of these receptors have beenshown to contribute to pathological manifestations of various diseases(Vassalli, 1992; Nagata and Golstein, 1995). Identifying the moleculesthat participate in the signaling activity of the receptors, and findingways to modulate the activity of these molecules, could direct newtherapeutic approaches. In view of the suspected central role of MACHαin Fas/APO1- and TNF-mediated toxicity, it seems particularly importantto design drugs that can block the proteolytic function of MACHα, as wasdone for some other proteins of the CED3/ICE family (Thornberry et al.,1994; Miller et al., 1995; Mashima et al., 1995; Milligan et al., 1995;Enari et al., 1995; Los et al., 1995). The unique sequence features ofthe CED3/ICE homolog within MACHA molecules could permit the design ofdrugs that would specifically affect its activity. Such drugs couldprovide protection from excessive immune-mediated cytotoxicity involvingMACHα, without interfering with the physiological cell-death processesin which other members of the CED3/ICE family are involved.

Other aspects of the invention will be apparent from the followingexamples.

The invention will now be described in more detail in the followingnon-limiting examples and the accompanying drawings.

It should also be noted that the procedures of: i) two-hybrid screen andtwo-hybrid β-galactosidase expression test; (ii) induced expression,metabolic labeling and immunoprecipitation of proteins; (iii) in vitrobinding; (iv) assessment of the cytotoxicity; and (v) Northern andsequence analyses, as set forth in Examples 1 (see also Boldin et al.,1995b) and 2 below, with respect to MORT-1 and a MORT-1 binding protein,are equally applicable (with some modifications) for the correspondingisolation, cloning and characterization of MACH and its isoforms. Theseprocedures are thus to be construed as the full disclosure of the sameprocedures used for the isolation, cloning and characterization of MACHin accordance with the present invention, as detailed in Example 3below.

EXAMPLE 1 Cloning and Isolation of the MORT-1 Protein Which Binds to theIntracellular Domain of the FAS-R

(i) Two-hybrid Screen and Two-hybrid β-Galactosidase Expression Test

To isolate proteins interacting with the intracellular domain of theFAS-R, the yeast two-hybrid system was used (Fields and Song, 1989).Briefly, this two-hybrid system is a yeast-based genetic assay to detectspecific protein-protein interactions in vivo by restoration of aeukaryotic transcriptional activator such as GAL4 that has two separatedomains, a DNA binding and an activation domain, which domains whenexpressed and bound together to form a restored GAL4 protein, is capableof binding to an upstream activating sequence which in turn activates apromoter that controls the expression of a reporter gene, such as lacZor HIS3, the expression of which is readily observed in the culturedcells. In this system, the genes for the candidate interacting proteinsare cloned into separate expression vectors. In one expression vector,the sequence of the one candidate protein is cloned in phase with thesequence of the GAL4 DNA-binding domain to generate a hybrid proteinwith the GAL4 DNA-binding domain, and in the other vector, the sequenceof the second candidate protein is cloned in phase with the sequence ofthe GAL4 activation domain to generate a hybrid protein with theGAL4-activation domain. The two hybrid vectors are then co-transformedinto a yeast host strain having a lacZ or HIS3 reporter gene under thecontrol of upstream GAL4 binding sites. Only those transformed hostcells (cotransformants) in which the two hybrid proteins are expressedand are capable of interacting with each other, will be capable ofexpressing the reporter gene. In the case of the lacZ reporter gene,host cells expressing this gene will become blue in color when X-gal isadded to the cultures. Hence, blue colonies are indicative of the factthat the two cloned candidate proteins are capable of interacting witheach other.

Using this two-hybrid system, the intracellular domain, FAS-IC, wascloned, separately, into the vector pGBT9 (carrying the GAL4 DNA-bindingsequence, provided by CLONTECH, USA, see below), to create fusionproteins with the GAL4 DNA-binding domain. For the cloning of FAS-R intopGBT9, a clone encoding the full-length cDNA sequence of FAS-R (WO9531544) was used from which the intracellular domain (IC) was excisedby standard procedures using various restriction enzymes and thenisolated by standard procedures and inserted into the pGBT9 vector,opened in its multiple cloning site region (MCS), with the correspondingsuitable restriction enzymes. It should be noted that the FAS-IC extendsfrom amino acid residues 175-319 of the intact FAS-R, this portioncontaining residues 175-319 being the FAS-IC inserted into the pGBT9vector.

The above hybrid (chimeric) vector was then cotransfected together witha cDNA library from human HeLa cells cloned into the pGAD GH vector,bearing the GAL4 activating domain, into the HF7c yeast host strain (allthe above-noted vectors, pGBT9 and pGAD GH carrying the HeLa cell cDNAlibrary, and the yeast strain were purchased from Clontech Laboratories,Inc., USA, as a part of MATCHMAKER Two-Hybrid System, #PT1265-1). Theco-transfected yeasts were selected for their ability to grow in mediumlacking Histidine (His⁻ medium), growing colonies being indicative ofpositive transformants. The selected yeast clones were then tested fortheir ability to express the lacZ gene, i.e., for their LACZ activity,and this by adding X-gal to the culture medium, which is catabolized toform a blue colored product by β-galactosidase, the enzyme encoded bythe lacZ gene. Thus, blue colonies are indicative of an active lacZgene. For activity of the lacZ gene, it is necessary that the GAL4transcription activator be present in an active form in the transformedclones, namely that the GAL4 DNA-binding domain encoded by the abovehybrid vector be combined properly with the GAL4 activation domainencoded by the other hybrid vector. Such a combination is only possibleif the two proteins fused to each of the GAL4 domains are capable ofstably interacting (binding) to each other. Thus, the His⁺ and blue(LACZ⁺) colonies that were isolated are colonies which have beencotransfected with a vector encoding FAS-IC and a vector encoding aprotein product of human HeLa cell origin that is capable of bindingstably to FAS-IC.

The plasmid DNA from the above His⁺, LACZ⁺ yeast colonies was isolatedand electroporated into E. coli strain HB101 by standard proceduresfollowed by selection of Leu⁺ and Ampicillin resistant transformants,these transformants being the ones carrying the hybrid pGAD GH vectorwhich has both the Amp^(R) and Leu2 coding sequences. Such transformantstherefore are clones carrying the sequences encoding newly identifiedproteins capable of binding to the FAS-IC. Plasmid DNA was then isolatedfrom these transformed E. coli and retested by:

(a) retransforming them with the original FAS-R intracellular domainhybrid plasmid (hybrid pGTB9 carrying the FAS-IC) into yeast strain HF7as set forth hereinabove. As controls, vectors carrying irrelevantprotein encoding sequences, e.g., pACT-lamin or pGBT9 alone were usedfor cotransformation with the FAS-IC-binding protein (i.e.,MORT-1)-encoding plasmid. The cotransformed yeasts were then tested forgrowth on His⁻ medium alone, or with different levels of3-aminotriazole; and

(b) retransforming the plasmid DNA and original FAS-IC hybrid plasmidand control plasmids described in (a) into yeast host cells of strainSFY526 and determining the LACZ⁺ activity (effectivity of β-galformation, i.e., blue color formation).

The results of the above tests revealed that the pattern of growth ofcolonies in His- medium was identical to the pattern of LACZ activity,as assessed by the color of the colony, i.e., His⁺ colonies were alsoLACZ⁺. Further, the LACZ activity in liquid culture (preferred cultureconditions) was assessed after transfection of the GAL4 DNA-binding andactivation-domain hybrids into the SFY526 yeast hosts which have abetter LACZ inducibility with the GAL4 transcription activator than thatof the HF7 yeast host cells.

Using the above procedure, a protein called previously designated, andnow referred to as MORT-1 for “Mediator of Receptor-induced Toxicity”,was identified, isolated and characterized.

Furthermore, it should also be mentioned that in a number of the abovetwo-hybrid β-galactosidase expression tests, the expression ofβ-galactosidase was also assessed by a preferred filter assay. In thescreening, five of about 3×10⁶ cDNAs were found to contain the MORT-1insert. The so-isolated cloned MORT-1 cDNA inserts were then sequencedusing standard DNA sequencing procedures. The amino acid sequence ofMORT-1 (SEQ ID NO:2) was deduced from the DNA sequence. Residuenumbering in the proteins encoded by the cDNA inserts are as in theSwiss-Prot data bank. Deletion mutants were produced by PCR, and pointmutants by oligonucleotide-directed mutagenesis (Current Protocols inMolec. Biol., 1994).

(ii) Induced Expression, Metabolic Labeling and Immunoprecipitation ofProteins

MORT-1, N-linked to the FLAG octapeptide (FLAG-MORT-1; Eastman Kodak,New Haven, Conn., USA), Fas-IC, FAS-R, p55-R, a chimera comprised of theextracellular domain of p55-R (amino acids 1-168) fused to thetransmembrane and intracellular domain of FAS-R (amino acids 153-319),and the luciferase cDNA which serves as a control, were expressed inHeLa cells. Expression was carried out using a tetracycline-controlledexpression vector, in a HeLa cell clone (HtTA-1) that expresses atetracycline-controlled transactivator (Gossen and Bujard, 1992; seealso Boldin et al., 1995). Metabolic labeling with [³⁵S]methionine and[³⁵S]cysteine (DUPONT, Wilmington, Del., USA and Amersham,Buckinghamshire, England) was performed 18 hours after transfection, bya further 4 h incubation at 37° C. in Dulbecco's modified Eagle's mediumlacking methionine and cysteine, but supplemented with 2% dialyzed fetalcalf serum. The cells were then lysed in RIPA buffer (10 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS and 1 mM EDTA) andthe lysate was precleared by incubation with irrelevant rabbit antiserum(3 μl/ml) and Protein G Sepharose beads (Pharmacia, Uppsala, Sweden; 60μl/ml). Immunoprecipitation was performed by 1 h incubation at 4° C. of0.3 ml aliquots of lysate with mouse monoclonal antibodies (5μl/aliquot) against the FLAG octopeptide (M2; Eastman Kodak), p55-R (#18and #20; Engelmann et al., 1990), or FAS-R (ZB4; Kamiya Southand Oaks,Calif., USA), or with isotype matched mouse antibodies as a control,followed by a further 1 h incubation with Protein G Sepharose beads (30μl/aliquot).

(iii) In vitro Binding

Glutathione S-transferase (GST) fusions with the wild type or a mutatedFas-IC were produced and adsorbed to glutathione-agarose beads; seeBoldin et al., 1995; Current Protocols in Molecular Biology, 1994;Frangioni and Neel, 1993). Binding of metabolically-labeled FLAG-MORT-1fusion protein to GST-Fas-IC was assessed by incubating the beads for 2h at 4° C. with extracts of HeLa cells, metabolically labeled with[³⁵S]methionine (60 μCi/ml), that express FLAG-MORT-1. The extracts wereprepared in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,0.1% NP-40, 1 mM dithiotreitol, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 20 μg/ml Aprotonin, 20 μg/ml Leupeptin, 10 mM sodium fluorideand 0.1 mM sodium vanadate (1 ml per 5×10⁵ cells).

(iv) Assessment of the Cytotoxicity Triggered by Induced Expression ofMORT-1

MORT-1, Fas-IC, p55-IC and luciferase cDNAs were inserted into atetracycline-controlled expression vector and transfected to HtTA-1cells (a HeLa cell line) (Gossen and Bujard, 1992) together with thesecreted placental alkaline phosphatase cDNA, placed under control ofSV40 promoter (the pSBC-2 vector, Dirks et al., 1993). Cell death wasassessed 40 hours after transfection, either by the neutral-red uptakeassay (Wallach, 1984) or, for assessing specifically the death in thosecells that express the transfected cDNAs, by determining the amounts ofplacental alkaline phosphatase (Berger et al., 1988) secreted to thegrowth medium at the last 5 hours of incubation.

In another set of experiments to analyze the region of the MORT-1protein involved in the binding to the FAS-IC, the following proteinswere expressed transiently in HeLa cells that contain atetracycline-controlled transactivator (HtTA-1), using atetracycline-controlled expression vector (pUHD10-3): Human FAS-R alone;Human FAS-R as well as the N-terminal part of MORT-1 (amino acids 1-117,the “MORT-1 head”); Human FAS-R as well as the C-terminal part ofMORT-1, which contains its ‘death domain’ homology region (amino acids130-245, the “MORT-1 DD”); FLAG-55.11 (amino acids 309-900 of protein55.11 fused at the N-terminus to the FLAG octapeptide, the protein 55.11being a p55-IC-specific binding protein. Twelve hours aftertransfection, the cells were trypsinized and re-seeded at aconcentration of 30,000 cells/well. After 24 hrs further incubation, thecells were treated for 6 hrs with a monoclonal antibody against theextracellular domain of FAS-R (monoclonal antibody CH-11, Oncor,Gaithersburg, Md., USA) at various concentrations (0.001-10 μg/mlmonoclonal antibody), in the presence of 10 μg/ml cycloheximide. Cellviability was then determined by the neutral-red uptake assay and theresults were presented in terms of % viable cells as compared to cellsthat had been incubated with cycloheximide alone (in the absence ofanti-FAS-R monoclonal antibody CH-11).

(v) Northern and Sequence Analyses

Poly A⁺ RNA was isolated from total RNA of HeLa cells (Oligotex-dT mRNAkit. QIAGEN, Hilden, Germany). Northern analysis using the MORT-1 cDNAas a probe was performed by conventional methods (see Boldin et al.,1995). The nucleotide sequence of MORT-1 was determined in bothdirections by the dideoxy chain termination method.

Sequence analysis of the MORT-1 cDNA cloned by the two-hybrid procedureindicated that it encodes a novel protein. Applying the two-hybrid testfurther to evaluate the specificity of the binding of this protein(MORT-1 for “Mediator of Receptor-induced Toxicity”) to Fas-IC, and todefine the particular region in Fas-IC to which it binds, led to thefollowing findings (FIG. 1): (a) The MORT-1 protein binds both to humanand to mouse. Fas-IC, but not to several other tested proteins,including three receptors of the TNF/NGF receptor family (p55 and p75TNF receptors and CD40); (b) Replacement mutations at position 225 (Ile)in the ‘death domain’ of FAS-R, shown to abolish signaling both in vitroand in vivo (the lpr^(cg) mutation (Watanabe-Fukunaga et al., 1992; Itohand Nagata, 1993), also prevents binding of MORT-1 to the FAS-IC; (c)The MORT-1 binding-site in FAS-R occurs within the ‘death domain’ ofthis receptor; and (d) MORT-1 binds to itself. This self-binding, andthe binding of MORT-1 to FAS-R involve different regions of the protein:A fragment of MORT-1 corresponding to residues 1-117 binds to thefull-length MORT-1, but does not bind to itself nor to the FAS-IC.Conversely, a fragment corresponding to residues 130-245 binds to FAS-R,yet does not bind to MORT-1 (FIG. 1). Furthermore, it is apparent fromthe results in FIG. 1 that the ‘death domain’ region of FAS-R iscritical for FAS-IC self-association, as is the ‘death domain’ region ofp55-R for p55-IC self-association. The deletions on both sides of these‘death domains’ does not affect the self-association ability thereofwhile, however, a deletion within these ‘death domains’ does affect theself-association. In the case of MORT-1, the binding of MORT-1 to FAS-ICis also dependent upon the complete (full) ‘death domain’ of FAS-R,while however, it is also not dependent on the regions outside of theFAS-R ‘death domain’ region for FAS-IC binding.

In FIG. 1, there is depicted the interaction of the proteins encoded bythe Gal4 DNA binding domain and activation-domain constructs (pGBT9 andPGAD-GH) within transfected SFY526 yeasts as assessed by β-galactosidaseexpression filter assay. The DNA-binding-domain constructs included fourconstructs of the human Fas-IC, four constructs of the mouse Fas-ICincluding two full-length constructs having Ile to Leu or Ile to Alareplacement mutations at position 225 (I225N and I225A, respectively),and three MORT-1 constructs, all of which are shown schematically on theleft hand side of FIG. 1. The activation-domain constructs includedthree MORT-1 constructs, the MORT-1 portion being as in theDNA-binding-domain constructs; and a full-length human Fas-IC construct,the Fas-IC portion being the same as in the above DNA-binding domainconstruct. The intracellular domains of human p55 TNF receptor (p55-ICresidues 206-426), human CD40 (CD40-IC, residues 216-277) and human p75TNF receptor (p75-IC, residues 287-461) as well as lamin, cyclin D and“empty” Gal4 (pGBT9) vectors served as negative controls in the form ofDNA-binding domain constructs. SNF-1 and SNF4 served as positivecontrols in the form of DNA-binding-domain (SNF1) and activation-domain(SNF4) constructs. “Empty” Gal4 vectors (pGAD-GH) also served asnegative controls in the form of activation domain constructs. Thesymbols “++” and “+” denote the development of strong color within 30and 90 min of the assay, respectively; and “−” denotes no development ofcolor within 24 h. Combinations for which no score is given have notbeen tested.

Expression of MORT-1 molecules fused at their N terminus with the FLAGoctapeptide (FLAG-MORT-1) yielded in HeLa cells proteins of fourdistinct sizes—about 27, 28, 32, and 34 kD. The interaction of MORT-1with Fas-IC in vitro was observed by performing an immunoprecipitate ofproteins from extracts of HeLa cells transfected with the FLAG-MORT-1fusion protein or with luciferase cDNA as a control, theimmunoprecipitation being performed with anti-FLAG antibody (αFLAG). Theinteraction in vitro was also demonstrated between MORT-1 and FAS-ICwherein MORT-1 is in the form of [³⁵S] methionine-metabolically labeledFLAG-MORT-1 fusion proteins obtained from extracts of transfected HeLacells and FAS-IC is in the form of human and mouse GST-FAS-IC fusionproteins including one having a replacement mutation at position 225 inFAS-IC, all of which GST-FAS-IC fusion proteins were produced in E.coli. The GST-fusion proteins were attached to glutathione beads beforeinteraction with the extracts containing the MORT-1-FLAG fusion proteinfollowing this interaction, SDS-PAGE was performed. Thus, the in vitrointeraction was evaluated by assessing, by autoradiography followingSDS-PAGE, the binding of [³⁵S] metabolically labeled MORT-1, produced intransfected HeLa cells as a fusion with the FLAG octapeptide(FLAG-MORT-1), to GST, GST fusion with the human or mouse Fas-IC(GST-huFas-IC, GST-mFas-IC) or to GST fusion with Fas-IC containing aIle to Ala replacement mutation at position 225. It was shown that allfour FLAG-MORT-1 proteins showed ability to bind to Fas-IC uponincubation with a GST-Fas-IC fusion protein. As in the yeast two-hybridtest (FIG. 1), MORT-1 did not bind to a GST-Fas-IC fusion protein with areplacement at the lpr^(cg) mutation site (I225A).

The proteins encoded by the FLAG-MORT-1 cDNA showed also an ability tobind to the intracellular domain of FAS-R, as well as to theintracellular domain of FAS-R chimera whose extracellular domain wasreplaced with that of p55-R (p55-FAS), when co-expressed with thesereceptors in HeLa cells. In this case, interaction of MORT-1 with FAS-ICin transfected HeLa cells, i.e., in vivo, as observed withimmunoprecipitates of various transfected HeLa cells demonstrated the invivo interaction and specificity of the interaction between MORT-1 andFAS-IC in cells co-transfected with constructs encoding these proteins.Thus, FLAG-MORT-1 fusion protein was expressed and metabolically labeledwith [³⁵S] cystein (20 μCi/ml) and [³⁵S]methionine (40 μCi/ml) in HeLacells, alone, or together with human FAS-R, FAS-R chimera in which theextracellular domain of FAS-R was replaced with the corresponding regionin the human p55-R (p55-FAS), or the human p55-R, as negative control.Cross immunoprecipitation of MORT-1 with the co-expressed receptor wasperformed using various specific antibodies. The results indicated that,FLAG-MORT-1 is capable of binding to the intracellular domain of FAS-R,as well as to the intracellular domain of a FAS-R-p55-R chimera havingthe extracellular domain of p55-R and the intracellular domain of FAS-R,when co-expressed with these receptors in the HeLa cells. Further,immunoprecipitation of FLAG-MORT-1 from extracts of the transfectedcells also resulted in precipitation of the co-expressed FAS-R or theco-expressed p55-FAS chimera. Conversely, immunoprecipitation of thesereceptors resulted in the coprecipitation of the FLAG-MORT-1.

Northern analysis using the MORT-1 cDNA as probe revealed a singlehybridizing transcript in HeLa cells. In a Northern blot in which polyA⁺ RNA (0.3 μg) from transfected cells was hybridized with MORT-1 cDNA,the size of the RNA transcript (about 1.8 kb) was found to be close tothe size of the MORT-1 cDNA (about 1702 nucleotides).

In sequence analysis, the cDNA was found to contain an open readingframe of about 250 amino acids. FIG. 2 depicts the preliminarynucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2)of MORT-1 in which the ‘death domain’ motif is underlined, as is apossible start Met residue (position 49; bold, underlined M) and thetranslation stop codon (the asterik under the codon at position769-771). This ‘death domain’ motif shares homology with the known p55-Rand FAS-R ‘death domain’ motifs (p55DD and FAS-DD). In order todetermine the precise C-terminal end of MORT-1 and to obtain evidenceconcerning the precise N-terminal (initial Met residue) end of MORT-1,additional experiments were carried out as follows:

Using the methods described above, a number of constructs encodingMORT-1 molecules fused at their N-terminus with the FLAG octapeptide(FLAG-MORT-1) were constructed and expressed in HeLa cells withmetabolic labeling of the expressed proteins using ³⁵S-cysteine and³⁵S-methionine. The MORT-1-FLAG molecules were encoded by the followingcDNAs containing different portions of the MORT-1-encoding sequence:

i) The FLAG octapeptide cDNA linked to the 5′ end of the MORT-1 cDNAfrom which nucleotides 1-145 of SEQ ID NO:1 (see FIG. 2) have beendeleted;

ii) The FLAG octapeptide cDNA linked to the 5′ end of the MORT-1 fulllength cDNA;

iii) The FLAG octapeptide cDNA linked to the 5′ end of the MORT-1 cDNAfrom which nucleotides 1-145 as well as nucleotides 832-1701 of SEQ IDNO:1 (FIG. 2) have been deleted and the codon GCC at position 142-144was mutated to TCC to prevent start of translation at this site.

Following expression of the above FLAG-MORT-1 fusion products,immunoprecipitation was carried out as mentioned above, using eitheranti-FLAG monoclonal antibodies (M2) or as a control, anti-p75 TNF-Rantibodies (#9), followed by SDS-PAGE (10% acrylamide) andautoradiography. The results of the analysis with the above FLAG-MORT-1fusion products confirmed (validated) the C-terminal end of MORT-1 andhave provided evidence that the N-terminal end of MORT-1 may be atposition 49 of the sequence in FIG. 2.

Indeed, it has been shown by additional expression experiments of MORT-1without the FLAG octapeptide fused to its 5′-end, that Met⁴⁹ serves asan effective site of translation initiation.

A search conducted in the ‘Gene Bank’ and ‘Protein Bank’ DataBasesrevealed that there is no sequence corresponding to that of the aboveisolated MORT-1 sequence. Thus, MORT-1 represents a new FAS-IC-specificbinding protein.

High expression of p55-IC results in triggering of a cytocidal effect(Boldin et al., 1995). The expression of Fas-IC in HeLa cells also hassuch an effect, though to a lower extent, which could be detected onlywith the use of a sensitive assay. The ligand independent triggering ofcytocidal effects in cells transfected with MORT-1, as well as humanp55-IC and FAS-IC, was thus analyzed. The effect of transient expressionof MORT-1, human Fas-IC, human p55-IC, or. luciferase that served as acontrol, on the viability of HeLa cells was assessed using atetracycline-controlled expression vector. Cell viability was evaluated40 min after transfecting these cDNAs either in the presence or absenceof tetracycline (1 μg/ml, to block expression), together with a cDNAencoding the secreted placental alkaline phosphatase. Cell viability wasdetermined either by the neutral red uptake assay or, for determiningspecifically the viability of those particular cells that express thetransfected DNA, by measuring the amounts of placental alkalinephosphatase secreted to the growth medium.

The above analysis revealed that the expression of MORT-1 in HeLa cellsresulted in significant cell death, greater than that caused by FAS-ICexpression. These cytotoxic effects of all of p55-IC, FAS-IC and MORT-1seem to be related to the ‘death domain’ regions, present in all ofthese proteins, which ‘death domains’ have a propensity toself-associate, and thereby possibly prompting the cytotoxic effects.

In view of the above mentioned characteristics of MORT-1, namely, thespecific association of MORT-1 with that particular region in FAS-Rwhich is involved in cell death induction, and the fact that even aslight change of structure in that region, which prevents signaling (thelpr^(cg) mutation) abolishes also the binding of MORT-1, indicates thatthis protein plays a role in the signaling or triggering of cell death.This notion is further supported by the observed ability of MORT-1 totrigger by itself a cytocidal effect. Thus, MORT-1 may function as (i) amodulator of the self-association of FAS-R by its own ability to bind toFAS-R as well as to itself, or (ii) serve as a docking site foradditional proteins that are involved in the FAS-R signaling, i.e.,MORT-1 may be a ‘docking’ protein and may therefore bind other receptorsbesides FAS-R, or (iii) constitutes part of a distinct signaling systemthat interacts with FAS-R signaling.

In order to further analyze the regions of MORT-1 involved in FAS-ICbinding and modulation of the FAS-R-mediated cellular effects(cytotoxicity), the above-mentioned experiments were carried out, usingvectors encoding portions of MORT-1 (the ‘MORT-1 head’, amino acids1-117 and the ‘MORT-1 dd’, amino acids 130-245) (separately), with avector encoding the human FAS-R for co-transfections of HeLa cells. Inthese experiments, the various proteins and combinations of proteinswere expressed transiently in HeLa cells that contain atetracycline-controlled transactivator (HtTA-1) by inserting thesequences encoding the proteins into a tetracycline-controlledexpression vector pUHD10-3. Control transfections employed vectorsencoding only the FAS-R and vectors encoding the FLAG-55.11 fusionprotein (the 55.11 protein being a p55-IC-specific binding protein ofwhich a portion containing amino acids 309-900 was fused (at itsN-terminal) to the FLAG octapeptide).

Following the transfection and incubation periods, the transfected cellswere treated with various concentrations of an anti-FAS-R monoclonalantibody (CH-11) which binds specifically to the extracellular domain ofFAS-R expressed by cells. This binding of anti-FAS-R antibody inducesthe aggregation of the FAS-R at the cell surface (much like the FAS-Rligand) and induces the intracellular signaling pathway mediated by theFAS-IC, resulting, ultimately, in cell death (FAS-R mediated cellcytotoxicity). The concentrations of the anti-FAS-R monoclonal antibody(CH-11) used were in the range of 0.01-10 μg/ml, usually concentrationssuch as 0.005; 0.05; 0.5 and 5 μg/ml. The cells were treated with theanti-FAS antibody in the presence of 10 μg/ml cycloheximide.

The results of the above analysis show that the expression of FAS-R inthe transfected cells conveys an increased sensitivity to the cytocidaleffects of the anti-FAS-R antibodies (compare “fas” to “55.11”).Further, the co-expression of the region in MORT-1 that contains the‘death domain’ homology region and FAS-R (“fas+MORT-1 dd) stronglyinterferes with FAS-induced (i.e. FAS-R mediated) cell death as would beexpected from the ability of the MORT-1 ‘death domain’ (DD) region tobind to the FAS-R ‘death domain’ (FAS-DD). Moreover, co-expression ofthe N-terminal part of MORT-1 and FAS-R (“fas+MORT1 he”) does notinterfere with FAS-R-mediated cell death and, if at all, somewhatenhances the cytotoxicity (i.e., slightly increased cell death).

Thus, the above results clearly indicated that the MORT-1 protein hastwo distinct regions as far as binding to the FAS-IC and mediation ofthe cell-cytotoxic activity of the FAS-IC are concerned.

These results therefore also provide a basis for the use of differentparts (i.e., active fragments or analogs) of the MORT-1 protein fordifferent pharmaceutical applications. For example, the analogs orfragments or derivatives thereof of the MORT-1 protein which containessentially only the C-terminal portion of MORT-1 inclusive of its‘death domain’ region may be used for inhibiting FAS-R-mediatedcytotoxic effects in FAS-R containing cells or tissues and therebyprotect these cells or tissues from the deleterious effects of the FAS-Rligand in cases such as, for example, acute hepatitis. Alternatively,the analogs or fragments or derivatives thereof of the MORT-1 proteinwhich contain essentially only the N-terminal portion of MORT-1 may beused for enhancing the FAS-R-mediated cytotoxic effects in FAS-Rcontaining cells and tissues, thereby leading to the enhanceddestruction of these cells or tissues when desired in cases such as, forexample, tumor cells and autoreactive T and B cells. As detailed hereinabove, the above uses of the different regions of MORT-1 may be carriedout using the various recombinant viruses (e.g., Vaccinia) to insert theMORT-1 region-encoding sequence into specific cells or tissues it isdesired to treat.

Furthermore, it is also possible to prepare and use various othermolecules such as, antibodies, peptides and organic molecules which havesequences or molecular structures corresponding to the above notedMORT-1 regions in order to achieve the same desired effects mediated bythese MORT-1 regions.

Moreover, MORT-1 may be utilized to specifically identify, isolate andcharacterize other proteins which are capable of binding to MORT-1(i.e., MORT-1-binding proteins); see Examples 2 and 3.

EXAMPLE 2 Isolation of a MORT-1 Binding Protein

(i) Two-hybrid Screen and Two-hybrid β-Galactosidase Expression Test

In a manner analogous to the procedure described in Example 1, using theintracellular domain of p55 TNF-R (p55 IC) and MORT-1 as baits, andscreening a human B-cell library, two cDNA clones were obtained, whichencode a protein product capable of binding to both MORT-1 and p55-IC.Both clones have identical nucleotide sequences at the 5′ end as shownin FIG. 3 (SEQ ID NO:3).

(ii) Binding Properties of the Newly Cloned cDNA, in Two Hybrid Screens

Using the above-mentioned yeast two-hybrid procedure, a constructcontaining the new MORT-1-binding protein cDNA was used as a “prey” towhich were added constructs of a number of “baits” in separatereactions, to determine the binding specificity of the MORT-1-bindingprotein encoded by this cDNA. These “baits” included constructs encodingMORT-1, portions of MORT-1 (MORT ‘head’, aa1-117, MORT ‘tail’, aa130-245), the p55 IC (206-426 p55) or portion thereof (the ‘deathdomain’, 326-426 p55; and others upstream of the ‘death domain’ i.e.206-326). The results are shown in Table 2.

TABLE 2 β-galactosidase Bait expression data MORT-1 +++ 130-245 MORT-1 +1-117 MORT-1 − 206-426 p55 +++ 326-426 p55 +++ 206-326 p55 − 206-308 p55− 206-345 p55 − p55 L35INI − Fas IC − 233-319 Fas − p75 IC − CD40 IC −pGBT10 − SNF1 − Cycline D − Lamin −

The above results of the two-hybrid β-galactosidase expression test ofthe binding of the clone to a large panel of baits confirmed that theprotein encoded by this clone binds specifically to the death domains ofboth the p55 TNF-R and MORT-1.

In general, the MORT-1 binding protein may be utilized directly tomodulate or mediate the MORT-1 associated effects on cells, or,indirectly, to modulate or mediate the FAS-R ligand effect on cells whenthis effect is modulated or mediated by MORT-1. The same holds true withrespect to other intracellular proteins or intracellular domains oftransmembrane proteins, as specifically demonstrated for the p55 TNF-Rherein.

MORT-1-binding proteins include those which bind specifically to theentire MORT-1 protein or those which bind to different regions of theMORT-1 protein, e.g., the above-noted N- and C-terminal regions ofMORT-1. The MORT-1-binding proteins which bind specifically to suchregions may be used to modulate the activity of these regions and hencethe specific activity of MORT-1 as determined by these regions.

EXAMPLE 3 Isolation and Characterization of the MACH Protein, AnotherMORT-1 Binding Protein

(i) Two-hybrid Screen, Two-hybrid β-Galactosidase Test, Sequencing andSequence Analysis

Using the procedure set forth in Examples 1 and 2 above, a full lengthconstruct encoding human MORT-1 protein was employed as a “bait” in theyeast two-hybrid system to isolate a cDNA clone encoding an additionalnew MORT-1 binding protein. This new protein was originally designatedMORT-2, and now redesignated and referred to as MACH (for MORT-1associated CED3 homolog), by virtue of its characteristics as detailedherein below.

This cDNA clone was sequenced by standard procedures as set forth inExamples 1 and 2 above. Sequence analysis by standard procedures andcomputer programs (see Examples 1 and 2) revealed that this cDNA has anovel sequence and encodes a novel protein (neither the DNA nor theamino acid sequences was found in GENBANK or PROTEIN BANK sequencedatabases). Further, the cDNA encoding MACH was revealed an ORF-B openreading frame which has strong homology to the region above (5′upstream) the ‘death domain’ motif of the MORT-1 protein (see Example1). In FIGS. 4A-C, the structure of that part of the MACH cDNA clonewhich contains ORF-B (235 aa residues; FIG. 4A); the deduced amino acidsequence (SEQ ID NO:5) of the MACH ORF-B (FIG. 4B); and the nucleotidesequence (SEQ ID NO:4) of the MACH cDNA molecule (FIG. 4C) are shown. InFIG. 4A, the hatched region of ORF-B is the region sharing high homologywith the region of MORT-1 upstream of the MORT-1 ‘death domain” motif,and this MACH ORF-B region of homology consisting of the amino acidresidues underlined in FIG. 4B.

The yeast two-hybrid test was further applied to evaluate thespecificity of binding of MACH to MORT-1, in particular, to define theregion in MORT-1 to which MACH binds, as well as to determine which ofthe MACH ORFs interacts with MORT-1, the procedures being as set forthherein above in Examples 1 and 2. Briefly, various MORT-1 and MACHconstructs were prepared for testing the interaction of the proteinsencoded by the Gal4 DNA-binding domain and activation domain constructswithin transfected SFY526 yeast cells as assessed by the β-galactosidaseexpression filter assay. The DNA-binding domain constructs were preparedin pGBT9 vectors and the activation domain constructs were prepared inpGAD-GM vectors. For the activation domain constructs, the full-lengthMACH cDNA was used (MACH), as was a construct encoding only the ORF-B(MACH B) region. Control activation domain constructs were thosecontaining the full-length MORT-1 coding sequence (MORT 1, positivecontrol) and those having no inserts, i.e., “empty” vectors (pGAD-GM).For the DNA-binding domain constructs, the full-length MORT-1 cDNA wasused (MORT 1), as were constructs encoding only the MORT-1 upstreamregion (MORT-1DD aa 130-245). Control DNA-binding domain constructs,which were constructed to determine also the specificity of the MACHbinding, included constructs encoding lamin (Lamin), residues 287-461 ofthe intracellular domain of the human p75 TNF-R (human p75 IC), cyclic D(cycD), SNF1, residues 206-426 of the intracellular domain of the humanp55 TNF-R (human p55 IC), the ‘death domain’ region of the intracellulardomain of the human Fas-R (human Fas DD), residues 216-277 of theintracellular domain of the human CD40 (human CD40 IC), vectors withoutinsert or “empty” pGBT9 vectors (pGBT9, negative control), and aconstruct encoding the ORF-B region of MACH (MACH B). In the assay, thedevelopment of color was determined, where the greater the colordevelopment, the greater the interaction between the constructs encodedby the DNA-binding domain and activation domain. Color development wasdepicted by symbols, where “+++” and “+” indicate the development of astrong color within 30 and 90 min. of the assay, respectively, andindicates the lack of development of color within 24 hrs. of the assay.In cases where interactions were not tested, no symbol was indicated.The results of the various interactions for the above case are set forthin Table 3, while the results of the various interactions of the MACHisoforms are depicted in FIG. 5.

TABLE 3 DNA-Binding Domain DOMAIN HYBRID Hybrid MACH MACH B MORT 1pGAD-GH MORT-1 +++ +++ +++ −−− Bindinq region in MORT-1 MORT1 (-117)MORT1DD (130-245) −−− −−− Specificity tests Lamin −−− −−− human p75 IC−−− cyc D SNF1 human p55 IC human FAS DD −−− human CD40 IC −−− pGBT9 −−−MACH B + + −−−

Thus, as arises from the results shown in Table 3 above, it is apparentthat:

(a) MACH binds to MORT-1 in a very strong and specific manner;

(b) The MACH binding site in MORT-1 occurs before (upstream of) the‘death domain’ motif in MORT-1, i.e., it is in the region of MORT-1defined by aa 1-117 of MORT-1;

(c) The ORF-B region of MACH is the MORT-1-interacting region of theMACH protein; and

(d) The MACH ORF-B region is capable of self-association.

(ii) Cell-cytotoxic Effects Mediated by the Self-association Capabilityof the MACH Protein

The observation that MACH can self-associate, in particular, that theORF-B region of MACH self-associates and the previous correlationbetween self-association and cell-cytotoxicity as observed for theintracellular domains of p55 TNF-R and FAS-R, and as observed for MORT-1(see Example 1), suggested that MACH self-association may also beinvolved in cell-cytotoxicity.

In order to test this possibility, constructs encoding MACH wereprepared with a tetracycline-controlled expression vector (for detailssee Example 1). These constructs were used to transfect HeLa cells inwhich the vectors were transiently expressed. Besides the MACHconstructs, other control constructs were used to evaluate the effect oftransient expression on the viability of the HeLa cells to which theeffect of the MACH constructs could be compared. These other constructsincluded MORT-1, human FAS-IC and luciferase (Luc). In addition,co-transfection of the HeLa cells was also tested by using MORT-1 andMACH constructs to determine what effects the interaction between theseproteins would cause. After transfection the HeLa cells were incubatedand cell viability was evaluated 48 hrs. after transfection either inthe presence or the absence of tetracycline (1 μg/ml) to blockexpression. Cell viability was determined by the neutral red uptakeassay.

The results are shown in FIG. 6, which depicts graphically theligand-independent triggering of cytocidal effects in cells transfectedwith MACH in comparison to cells transfected with constructs encodingthe other proteins as well as cotransfected cells (MORT1+MACH). Theresults are shown as the cell viability in OD units at 540 nm for eachconstruct, wherein for each construct a hatched bar indicates incubationof cells following transfection in the absence of tetracycline, and afilled bar indicates incubation of the transfected cells in the presenceof tetracycline.

From the results shown in FIG. 6, it is apparent that MACH induces adramatic cytotoxic effect in HeLa cells, i.e., the inducedoverexpression of MACH cDNA in HeLa cells, resulting in a dramaticcytotoxic effect. This cytotoxic effect is likely to be related to theself-association capability of MACH.

(iii) Northern Analysis

Using well-known procedures (see Example 1), Northern analysis ofseveral cell lines was carried out using the MACH cDNA as a probe. Theresults of this analysis show that in a large number of cell lines, inparticular, CEM, Raji, Daudi, HeLa, Alexander, Juskat and A673 celllines, there exist two hybridizing transcripts of approximately 3.2 kbin size.

In view of the above, the MACH protein, particularly the MACHβ1 protein(ORF-B of MACH) may be utilized directly to modulate or mediate theMORT-1 associated effects on cells, or, indirectly, to modulate ormediate the FAS-R ligand effect on cells when this effect is modulatedor mediated by MORT-1. The fact that MACH binds specifically to theupstream region of MORT-1 and shares homology with MORT-1 provides for aspecific way in which MACH or MACH ORF-B may be used to modulate thisspecific region of MORT-1 and hence the specific activity of MORT-1determined by this upstream region. Further, MACH or MACH ORF-B may beused as a modulator or mediator of intracellular effects in an analogousway to MORT-1 itself (see above) by virtue of MACH's ability toself-associate and induce cell-cytotoxicity on its own.

Further analyses of the MACH protein and the DNA sequences encoding ithave been performed as set forth herein below. Further, it was revealedthat ORF-B of MACH represents but one of a number of MACH isoforms.Hence, the MACH protein and the DNA sequences encoding it have now beenrenamed, as will become apparent from the following.

(a) Two Hybrid Screen for Proteins that Bind to MORT-1 Reveals a NovelProtein which Shares a Sequence Motif with MORT-1:

As mentioned above, to identify proteins which participate in theinduction of cell death by MORT-1, the two-hybrid technique was used toscreen cDNA libraries for proteins that bind to MORT-1. A two-hybridscreen of a human B cell library (Durfee et al., 1993) using MORT-1 cDNAas bait yielded cDNA clones of MORT-1 itself, reflecting the ability ofthis protein to self-associate as well as clones of TRADD, to whichMORT-1 binds effectively (see Example 2). The screen also yielded cDNAclones of a novel sequence whose product specifically bound to MORT-1.The protein, which initially was called MACH, and later, after findingthat it occurs in multiple isoforms (see below), renamed MACHβ1, showedalso an ability to bind in a two hybrid test to itself, yet was unableto bind to FAS-R.

In FIG. 5, there is shown the results of the interaction of MORT-1 andMACH within transfected yeast cells. Briefly, MORT-1 and MACHβ1 andtheir deletion constructs, as well as MACHα1, a MACHα1 mutant in whichthe catalytic cysteine Cys₃₆₀ is replaced by Ser (MACHα1 (C360S)) andthe intracellular domain of human FAS-R (Fas-IC), were expressed withintransfected SFY526 yeast in Gal4 DNA binding domain and activationdomain constructs (pGBT9 and pGAD-GH). Their interaction was assessed bya β-galactosidase expression filter assay as described in Boldin et al.(1995b). The results are presented in terms of the time required for thedevelopment of strong color. ND indicates that the assay was not done.None of the inserts examined interacted with a number of tested negativecontrols, including the intracellular domains of human p55 TNF receptor,p75 TNF receptor and CD40, and lamin, cyclin D and ‘empty’ Gal4 vectors.MACHβ1 was cloned by two hybrid screening of a Gal4 AD-tagged human Bcell library (Durfee et al., 1993) for proteins that bind to MORT-1,using the HF7c yeast reporter strain. Except where otherwise indicated,all experimental procedures for the findings presented are as describedabove (see also Boldin et al., 1995). Deletion analysis showed thatMACHβ1 binds to the N-terminal part of MORT-1, which is involved in celldeath induction (Chinnaiyan et al. 1995). MACHβ1 also self-associated inthe transfected yeast. However, it did not bind to several controlproteins and unlike MORT-1 was unable to bind to FAS-R (FIG. 5).Expression of MACHβ1 molecules in mammalian cells yielded a 34 kDaprotein that bound to MORT-1 molecules co-expressed with it. It was alsoable to bind to a GST-MORT-1 fusion protein in vitro.

Comparison of the amino acid sequences in MACHβ1 and MORT-1 revealed ashared sequence motif (designated “Mort module”) in these two proteins,distinct from the death motif through which MORT-1 binds to FAS-R. Thismotif occurs once in MORT-1 and twice in MACHβ1. The same motif is foundalso in PEA-15, an astrocyte phosphoprotein of unknown function.Preliminary data suggest that the MORT motif is involved in the bindingof MACHβ1 (and of other MACH isoforms) to MORT-1.

FIG. 7A depicts the deduced amino acid sequence (SEQ ID NO:5) of MACHβ1.The two MORT modules are boxed and the C-termini of the two MACHβ1deletion mutants employed (FIG. 7) are denoted by asterisks. FIG. 7Bshows the sequence homology of the modules in MACHβ1 (designated MACH inFIG. 7B), MORT-1 and the PEA-15 gene (accession number X86809).Identical and similar residues are denoted by boxed and shaded areas,respectively.

FIG. 8 shows a diagrammatic representation of the death domain and MORTmodules and of the CED3/ICE homology region in Fas/APO1, MACHβ1 andMACHα1.

The region in MORT-1 that contains this ‘MORT module’ has been shown totake part in cell death induction by this protein (see Example 1 above).It has been shown also to contribute to, though not to suffice in, theself association of MORT-1 (see Example 1). As shown in FIG. 5, analysisof the binding properties of deletion constructs of MACHβ1 intransfected yeasts revealed similar involvement of the MORT modules inself-association of MACHβ1, as well as in its binding to MORT-1:Deletion constructs, in which the region below (downstream of) the MORTmodule was missing, were unable to bind to each other, yet maintainedthe ability to bind to the full length MORT-1 and to the full lengthMACHβ1. A further truncation in which part of the MORT module sequencewas also deleted, resulted in loss of the binding ability of theproteins. To further assess the involvement of the MORT modules in theseinteractions, deletion mutants of MACHβ1, fused with the FLAGoctapeptide (FLAG-MACHβ1), were expressed in HeLa cells and assessed fortheir binding in vitro to bacterial-producedglutathione-S-transferase-MORT-1 fusion protein (GST-MORT-1). As shownin FIGS. 9A-C, similarly to the binding observed in the yeast two-hybridtest, this in vitro binding was found to depend on interaction of theregion within MACHβ1 modules. FIGS. 9A and 9B show the results(autoradiograms) of the in vitro interaction of MACHβ1 and its deletionmutants with MORT-1. Briefly, ³⁵[S] metabolically labeled MACHβ1, MACHβ1fused at its N-terminus to the FLAG octapeptide (FLAG-MACHβ1),C-terminus truncation mutants of FLAG-MACHβ1, and, as a control,luciferase, were produced in transfected HeLa cells. Expression was doneusing a tetracycline-controlled expression vector, in a HeLa cell clone(HtTA-1) that expresses a tetracycline-controlled transactivator.

FIG. 9A shows the assessment of the expression of the proteins and theirmolecular sizes by immunoprecipitation from cell lysates, usinganti-FLAG antibody. The antibodies used are as follows: Rabbitanti-MACHβ1 and anti-MORT1 antisera were raised against GST-MACHβ1 andGST-MORT1 fusion proteins. Mouse monoclonal antibodies against the FLAGoctapeptide (M2) and against FAS/APO1 (CH11, Yonehara et al., 1989) werepurchased from Eastman Kodak and Oncor (Gaithersburg, Md.) respectively.Mouse monoclonal anti-HA epitope antibody (12CA5, Field et al., 1988)and anti-TNF antibody were produced in our laboratory according to theusual methods well known in the art. FIG. 9B shows affinity binding ofthe proteins to GST-MORT-1, adsorbed to glutathione-agarose beads (or,as a control, to GST or GST-fused to the intracellular domain ofFas-APO1). FIG. 9C shows the results of the immuno-precipitations of thevarious MORT-1 and MACH fusion constructs using the various specificantibodies.

(b) MACH Occurs in Multiple Isoforms:

Northern analysis using MACHβ1 cDNA as a probe revealed low abundanttranscript(s) of approximately 3 kb in size in several different celllines. Briefly, Northern blot analysis of total RNA (14 μg/lane) or polyA⁺RNA (2 μg) from several cell lines, using MACHβ1 cDNA as probe wasperformed. The cell lines examined, T47D, CEM, Raji, Daudi, HeLa,Alexander, Jurkat and A673, are all of human origin and were derivedfrom a ductal carcinoma of the breast, an acute lymphoblastic T cellleukemia, a Burkitt lymphoma, a Burkitt lymphoma, an epitheloidcarcinoma, a human hepatoma, an acute T cell leukemia and arhabdomyosarcoma, respectively. The rather diffuse shape of thehybridizing band on Northern blots suggested that these transcripts areof heterogeneous sizes ranging between 2.85 and 3.5 Kb. Both the amountsand the sizes of the transcripts varied among different human tissuesand were not correlated with the expression of MORT1 (Chinnaiyan et al.,1995) or of FAS/APO1 (Watanabe et al., 1992). cDNA prbes wereradiolabeled with the random-prime kit (Boehringer Mannheim) and appliedfor analysis of human multiple tissue blots (Clontech) according to themanufacturer's instructions. In the testis and skeletal muscle, forexample, MACH transcripts were barely detectable, even though thesetissues express significant amounts of MORT1. Conversely, restingperipheral blood mononuclear leukocytes, in which MORT1 expression isvery low, were found to express MACH at high levels. Lectin activationof the leukocytes results in a marked change in the size pattern of MACHtranscripts, along with an induction of MORT-1.

Exploring the nature of this size heterogeneity, cDNA libraries werescreened for transcripts that hybridize with the MACHβ1 cDNA probe.MACHα1 and MACHα2 were cloned from a Charon BS cDNA library derived fromthe mRNA of human thymus. The library was screened under stringentconditions with a MACHβ1 cDNA probe, labeled using a random-priming kit(Boehringer Mannheim). The other MACH isoforms were cloned by RT-PCR,performed on total RNA from Raji (MACHα1, α2, α3, β3, β4 and β5) andDaudi (MACHα2, β3, β4, and β5) human lymphoblastoid cells. Reversetranscriptase reaction was performed with an oligo-dT adapter primer(5′-GACTCGAGTCTAGAGTCGAC(T)₁₇-3′; SEQ ID NO:26) and the SuperScript IIreverse transcriptase (GIBCO-BRL), used according to the manufacturer'sinstructions. The first round of PCR was performed with the Expand LongTemplate PCR System (Boehringer Mannheim) using the following sense andantisense primers: 5′-AAGTGAGCAGATCAGAATTGAG-3′, corresponding tonucleotides 530-551 of the MACHβ1 cDNA (SEQ ID NO:4), and5′-GACTCGAGTCTAGAGTCGAC-3′ (SEQ ID NO:27), respectively. The secondround was performed with Vent polymerase (NEB) using the following senseand antisense nested primers: 5′GAGGATCCCCAAATGCAAACTGGATGATGAC-3′ (SEQID NO:28) and 5′-GCCACCAGCTAAAAACATTCTCAA-3′, (corresponding tonucleotides 962-939 of SEQ ID NO:4) of MACHβ1 cDNA, respectively. Toconfirm that MACHβ3 and MACHβ4 have initiation codons, a more 5′sequence of these isoforms from the RNA of Raji cells was cloned. TheRT-PCR reaction, performed using the oligo-dT adapter primer asdescribed above, was followed by two rounds of PCR (with Vent polymerase(NEB)) using the following sense and antisense oligonucleotides:5′-TTGGATCCAGATGGACTTCAGCAGAAATCTT-3′ (SEQ ID NO:29) and5′-ATTCTCAAACCCTGCATCCAAGTG-3′ (corresponding to nucleotides 946-923 ofSEQ ID NO:4) in MACHβ1. The latter oligonucleotide is specific to theβ-isoforms. Among the clones obtained in this way, those found tocontain the nucleotides encoding for the amino acids of ‘block 2’ (whosepresence distinguishes MACHβ3 and MACHβ4 from MACHβ1 and MACHβ2 asdiscussed below) were fully sequenced. Nucleotide sequences in allcloned isoforms were determined in both directions by the dideoxy-chaintermination method. Only partial cDNA clones of MACHα3 and MACHβ2 wereobtained. This screening revealed the existence of multiple isoforms ofMACH. The amino acid sequences of eight of these isoforms were studiedin detail. The results are illustrated diagrammatically in FIG. 12 andexemplified in FIG. 13 where the amino acid sequences of three of theisoforms are compared with known homologs.

FIG. 10 shows a diagrammatic representation of the various MACHisoforms. Coding regions are represented as boxed areas. The variousdomains within the coding regions are denoted by different shadings asfollows: the MORT modules (#); the three amino acid sequence blockswhich occur in different combinations in the isoforms. Positions of theresidues in the CED3/ICE homology region implicated in the catalyticactivity of ICE based on its X-ray crystal structure are shown. Thecatalytic cysteine residue is also indicated by a star (★). Those partsof the MACHα1 nucleotide sequence that are missing in the sequences ofother isoforms are indicated in the diagrams of the latter isoforms byV-shaped connecting lines. The lengths of these cDNA regions, whichprobably correspond to distinct exons, are indicated below the diagramof MACHα1. Lack of the 65 nucleotides which in MACHα1 encode for ‘block2’ causes alteration in MACHβ1 and MACHβ2 of the reading frame of thenucleotides that encode for ‘block 3’. In those isoforms, therefore,these nucleotides encode other amino acids which together constitutetheir unique C-terminal region. On the other hand, in MACHβ3 and MACHβ4the reading frame of block 3 is maintained, but absence of thenucleotides that encode the CED3/ICE region and part of the 3′ noncodingregion results in alteration of the reading frame of nucleotides furtherdownstream. Because of this alteration, the most 5′ part of thisnoncoding downstream region does encode 10 amino acids, which constitutethe C-terminal region unique to these two isoforms (hatched). Asindicated in the figure, only partial cDNA clones of MACHα3 and MACHβ2were obtained.

The isoforms were cloned from a human B cell cDNA library (MACHβ1), froma human thymus cDNA library (MACHα1 and α2) and from the mRNA of thehuman lymphoblastoid cells Raji (MACH2α1, α2, α3, β3, β4, and β5) andDaudi (MACHα2, β2, β3, β4, and β5). Cloning from the mRNA of the Rajiand Daudi cells was done by RT-PCR, using oligonucleotides correspondingto a 3′ noncoding region and to a sequence within the second MORT modulein MACHβ1. The starting codon of clones isolated in that way istherefore located within the second MORT module. The cDNA sequence andamino acid sequence of the MACH isoforms are presented in the sequencelisting and identified as follows in Table 4.

TABLE 4 MACH isoform cDNA Sequence Amino Acid Sequence MACHα1 SEQ IDNO:14 SEQ ID NO:7  MACHα2 SEQ ID NO:17 SEQ ID NO:18 MACHα3 SEQ ID NO:19SEQ ID NO:20 MACHβ1 SEQ ID NO:4  SEQ ID NO:5  MACHβ2 SEQ ID NO:21 SEQ IDNO:22 MACHβ3 SEQ ID NO:23 SEQ ID NO:8  MACHβ4 SEQ ID NO:24 SEQ ID NC:25MACHβ5 SEQ ID NO:33 SEQ ID NO:34

The sequences in the different isoforms relate to each other as follows:(a) All the MACH isoforms share a common 182-amino acid N-terminalregion which encompasses the MORT modules, yet vary carboxy terminally(3′ downstream) to these modules, as well as in their noncoding regions.(b) On the basis of their C terminal sequences, the isoforms fall intotwo subgroups: four isoforms defined as subgroup β, have differentC-termini due to alteration in the reading frame. Two (MACHβ1 AND β2)share the C-terminus found in the isoform initially cloned in thetwo-hybrid screen and two (MACHβ3 and β4) share a different C-terminus;three isoforms, defined as subgroup α, have a much longer C-terminalregion that closely resemble proteases of the CED3/ICE family (seebelow); (c) The regions extending between the MORT module region and theC terminal region that defines the subgroups varied from one isoform toanother. However, close examination showed that these intermediateregions consist of different combinations of the same three amino acidsequence blocks (blocks 1, 2 and 3). The variations of amino acidsequence among the different clones reflect two kinds of variations innucleotide sequence, that most likely occur by alternative splicing: (a)insertion or absence of either of two nucleotide sequences, one of 45nucleotides (nts) and the other of 65 nts, or of both, below thenucleotides encoding Lys184; (b) presence of an additional insert withinthe region which in MACHβ1 constitutes the 3′ noncoding part. Thesevariations affect both the reading frame and the length of the protein.

Part of the MACH isoforms encompass a CED3/ICE homolog. Data bank searchrevealed that the C terminal region of MACHα isoforms including block 3and the sequence extending downstream of it, closely resemble proteasesof the CED3/ICE family. FIG. 11 presents sequence comparison of thisregion in MACH and the various known human members of this family aswell as the Caenorhabditis elegans ced3 protein. CED3 (Ellis andHorvitz, 1986; Yuan et al., 1993), and the known human proteases of theCED3/ICE protease family: CPP32 (Fernandes-Alnemri et al., 1994), alsocalled apopain (Nicholson et al., 1995) and Yama (Tewari et al., 1995b),Mch2α (Fernandes-Alnemri et al., 1995), Ich-1 (Wang et al., 1994; thehuman homolog of the mouse Nedd2 protein, Kumar et al., 1994),ICE_(rel)II (Munday et al., 1995), ICE_(rel)II (Munday et al., 1995),also called TX and Ich2 (Faucheu et al., 1995; Kamens et al., 1995), andICE (Thornberry et al., 1992; Cerretti et al., 1992). FIG. 11 depictsschematically the colinear amino acid sequence alignment of the MACHisoforms and the various known members for the CED/ICE protease family.ohown are the amino acid sequences of MACHα1, MACHβ1, MACH⊖3 as well asof the Caenorhabditis elegans protease CED3, and of the known humanproteases of the CED3/ICE protease family.

The above C-terminal region of MACH most closely resembles CPP32 (with41% identity and 62% homology) and CED3 (with 34% identity and 56%homology). It shows a significantly lesser similarity to ICE (with 28%identity an 50% homology) and to its closely related homologsICE_(rel)II (also called TX and Ich2) and ICE_(rel)III. The similaritywas observed throughout almost the whole region starting from Tyr226within block 3, to the C terminus of the MACHα isoforms.

Two points of similarity are particularly notable:

(a) All known proteases of the CED3/ICE family cleave proteins at sitesdefined by the occurrence of Asp at the P1 position and a smallhydrophobic amino acid residue at P1′. Their specificity differs,though, with regard to other structural features of the substrate,including the nature of the residues at positions P2-P4. Accordingly,the active site residues involved in catalysis (corresponding to His237,Gly238 and Cys285 in ICE) and in the binding pocket for the carboxylateside chain of the P1 Asp (Argl79, Gln283, Arg341 and probably alsoSer347) are conserved among these proteases. As shown in FIG. 11, theseresidues (marked by shading of the residues and by full and emptycircles below the sequences) are also conserved in MACHal. There is oneexception, though—a conservative change of Ser to Thr at the sitecorresponding to Ser347 of ICE. Another slight, yet potentiallyimportant, sequence difference between MACHα isoforms and other membersof the protease family is an Arg to Gln replacement of the residuecorresponding to Arg286 of ICE. This residue, which is adjacent to theputative catalytic cysteine residue, is fully conserved in all otherCED3/ICE family members. Also part of the residues at the sites locatedclose to the substrate P2-P4 residues (marked by triangles below thesequences in FIG. 11) differ in the MACHA isoforms from those found inother CED3/ICE family members.

(b) Proteases of the CED3/ICE family contain sites of autocleavage.Several of the proteases are known indeed to be self-processed, and todepend on this processing for displaying maximal catalytic activity.Their fully bioactive form is composed of two noncovalently-associatedcleavage products, which differ in size (p20 and p17 in ICE; p17 and p12in CPP32, as marked by arrows in FIG. 11). Presence of potential sitesof autocleavage in other members of the family suggests that they aresubject to similar processing, and, similarly, depend on this processingfor exhibiting maximal activity. Such potential sites of autocleavageoccur in MACHα1 almost at the same locations as in the CPP32 (see shadedboxes in FIG. 11). The site corresponding to the N terminus of the p17subunit of CPP32 is located in the second conserved block of aminoacids, just a few amino acids upstream to the N terminus of theCED3/ICE-homology region (below Asp216). The site corresponding to thepoint of cleavage between the two subunits of CPP32 is located, as inall other members of the CED3/ICE family that are known to be cleaved, afew amino acids downstream to the catalytic cysteine residue (belowAsp374). This conservation suggests that the CED3/ICE homology region inMACHα1 is subject to proteolytic processing. The sizes of the twoexpected products of this cleavage are very close to that of the twosubunits of the processed CPP32 molecule.

(c)′ The CED3/ICE Homology Region in MACH has Proteolytic Activity.

To find out if the CED3/ICE homology region in MACHA possessesproteolytic activity, applicants expressed the region that extends fromthe potential cleavage site upstream to this region, between Asp216 andSer217, till the C terminus of the protein in bacteria, as a GST fusionprotein. The bacterial lysates were examined for ability to cleavefluorogenic peptide substrates, shown before to be cleaved by otherCED3/ICE homologs. Two substrate peptides were used: The first,Acetyl-Asp-Glu-Val-Asp-a-(4-Methyl-Coumaryl-7-Amide) (AC-DEVD-AMC),corresponds to a sequence in poly (ADP-ribose) polymerase (PARP), anuclear protein found to be cleaved in cells shortly after FAS-Rstimulation (Tewari et al., 1995b), as well as in other apoptopicprocesses (Kaufmann, 1989; Kaufmann et al. 1993; Lazebnik et al., 1994).This fluorogenic substrate is cleaved effectively by CPP32. The secondfluorogenic substrate, Acetyl-Tyr-Val-Ala-Asp-AMC (Ac-YVAD-AMC),corresponds to a substrate site for ICE in the IL-1β precursor. Thisfluorogenic substrate is cleaved by ICE. As shown in FIGS. 12A-F and13A-B, lysates of bacteria expressing the CED3/ICE homology region inMACHα1 cleaved effectively the PARP sequence-derived fluorogenicsubstrate. They had no measurable proteolytic activity, though, againstthe IL-1β-precursor sequence-derived fluorogenic substrate (controls),Ac-YVAD-AMC, which is an ICE cleavage site in IL-1β precursor(Thornberry et al., 1992). The proteolytic activity was blocked byiodacetic acid (5 mM), confirming that it is mediated by a thiolprotease. No cleavage was observed with lysates containing the GST-fusedMACH CED3/ICE-homology region in which the catalytic cysteine residueCys₃₆₀ was replaced by Ser. Also, lysates from bacteria that expressedthe full-length MACHα1 protein as a GST-fusion protein did not cleaveAc-DEVD-AMC, probably because of the absence of bacterial enzymescapable of processing the full-length molecule. Nor did cleavage occurwith lysates containing either of the two potential cleavage products ofthe CED3/ICE homology region.

FIGS. 12A-F and 13A show the kinetics of cleavage of the PARPsequence-derived fluorogenic substrate, Ac-DEVD-AMC (50 μM), by extractsof E. coli expressing a GST-fusion protein of the CED3/ICE homologyregion in MACHα1 (Ser217 through the C-terminus of the protein) ascompared to the lack of cleavage by extracts of bacteria expressingGST-fusion proteins of the full-length MACHα1 molecule or of either oneof the two potential proteolytic products of the CED3/ICE homologyregion (Ser217 till Asp374 and Asp374 through the C-terminus of theprotein).

It also shows the substrate concentration-dependence of the cleavage ofAc-DEVD-AMC, incubated for 180 min. with extracts of bacteria expressingthe MACHα1 CED3/ICE homology-region in fusion with GST (see FIG. 13B).No cleavage was observed in the presence of iodoacetic acid (5 mM). Theextracts had no activity on Ac-YVAD-AMC, a fluorogenic substratecorresponding to a substrate site for ICE in the IL-1β precursor.

Briefly, the GST-fusion proteins were produced in XL1-blue bacteriausing the pGEX3 expression vector. The bacteria were lysed by sonicationin a buffer containing 25 mM HEPES (pH 7.5), 0.1%3-[3-cholamidopropyl)dimethylamino]-1-propanesulfonate, 5 mM EDTA and 2mM DDT, followed by centrifugation at 16,000×g for 10 min. SDS-PAGEanalysis confirmed the presence of similar levels of the various fusionproteins in the lysates (not shown). 50 μl aliquots of the extracts (4mg/ml of total protein) were incubated at room temperature for theindicated periods in a 500 μl total volume reaction with the fluorogenicsubstrates, at the indicated concentrations. AMC release was measured byspectro-fluorometry at an excitation wavelength of 380 nm and anemission wavelength of 460 nm. The concentration of AMC was determinedfrom a standard curve. Both fluorogenic substrate peptides were obtainedfrom Peptide Institute Inc. (Osaka, Japan). Other CED3/ICE proteaseswere shown to exhibit full activity only after proteolytic processing,which occurs either by self-cleavage, or via their cleavage by otherproteases (reviewed in Kumar, 1995; Henkart, 1996). Applicants'observation that lysates of bacteria that express GST-MACHα1 moleculesdo not possess enzymatic activity, as opposed to the activity observedin lysates of bacteria that express the CED3/ICE homology region,suggests that processing is also required for MACHα activity. The way inwhich MACHα processing occurs within the mammalian cell, and how thisprocessing is brought about by FAS-R or p55-R triggering, is not known.MORT-1 has been shown to bind in cells to acitivated FAS-R together withsome other proteins (Kischkel et al., 1995). These proteins are likelyto include MACHα1 and other MACH isoforms. It seems plausible that thebinding of MORT-1 in association with MACHα to FAS-R brings togetherseveral MACH molecules, or induces conformational changes in them, andthat these changes either trigger autolytic processing of MACHα or makeMACHα susceptible to cleabage by other proteases. Stimulation of p55-Rmay trigger self-processing of MACHα in a similar, though less directmanner, by brining together several TRADD molecules, or inducing aconformational change in them, which in turn induces a change in thevormation or state of aggregation of MORT-1 and its associated MACHmolecule.

The substrate specificity of MACHα seems to be rather ‘death oriented’.Although it could cleave a substrate peptide corrsponding to a cleavagesite in the death substrate PARP (Ac-DEVD-AMC), MACHα showed noproteolytic activity towards a peptide corresponding to the site ofprocessing of the IL-1β precursor by ICE (Ac-YVAD-AMC). Identificationof the cellular proteins that serve as substrates for cleavage by MACHαwill elucidate the more downstream events in death induction by thisprotease. Likely substrates for MACHα cleavage are other members of theCED3/ICE family, like CPP32 and ICE. Some of these proteases are indeedprocessed after FAS-R or TNF receptor-triggering (Miura et al., 1995;Schlegel et al, 1996; Chinnaiyan et al., 1996). Perhaps proteases thatdo not belong the CED3/ICE family are also activate by MACHα, eitherdirectly or through the action of other CED3/ICE proteases. Involvementof multiple proteases in the cell death process is consistent with thereported ability of inhibitors of various proteases, includinginhibitors of serine proteases and an inhibitor of ICE cleavage as wellas antisense ICE cDNA, to protect cells from FAS-R and TNFreceptor-induced toxicity (Weitzen and Granger, 1980; Ruggiero et al.,1987; Enari et al., 1995; Los et al., 1995).

A variety of other enzymes, including phospholipases, sphingomyelinasesand protein kinases, may participate in cell death induciton by the TNFreceptors and FAS-R (see Eischen et al., 1994; Vandenabeele et al.,1995; Cifone et al., 1995 and references therein). Some of these enzymesmay become activated by the proteolytic cleavage initiated by MACHα. Italso seems possible, however, that at least part of these otherdeath-related activities are stimulated by distinct signaling routes,independently of MACHα stimulation. Involvement of more than onesignaling cascade in the induction of cell death, some common to p55-Rand Fas/APO1 and some induced by only one of them, would be consistentwith report on both shared and distinct features of cell death processesinduced by the two receptors (Grell et al., 1994; Schulze-Osthoff etal., 1994; Wong and Goeddel, 1994; Clement and Stamenkovic, 1994).

(d) MACHα1 Binds to MORT1 as Well as to MACHβ1:

To find out if MACHα1 can bind to MORT1, as does MACHβ1, the interactionof the proteins within transfected yeasts was first examined. MACHα1appeared to have a significant cytotoxic effect on the yeasts. Thiseffect was manifested in a marked decrease in the yield of colonies inyeasts that expressed the protein in the activation domain (AD) vector(whose expression level is higher than that of the DNA binding domain(DBD) vector). On the other hand, MACHβ1 in which the catalytic cysteineresidue, Cys₃₆₀, was replaced with Ser (MACHα1(C360S)) was not cytotoxicto either mammalian cells (see below), or yeast. Like MACHβ1, MACHα1(C360S) bound in tranfected yeast to MORT-1 and also to itself. It alsobound to MACHβ1. Also, yeast expressing the wild-type MACHα1 togetherwith MORT-1 or MACHβ1 exhibited interaction of the transfected proteins.The intensity of the lacZ-product color varied, however, among the yeastcolonies; in yeasts tranfected with MACHα1 in both the AD and the DBDvectors no color product was observed, probably because of the cytotoxiceffect of the wild-type MACHα1. Yet, in spite of this variation, yeastsexpressing MACHα1 either in combination with MORT1 or in combinationwith MACHβ1 scored clearly positive for interaction of the transfectedproteins. Unlike MACHβ1, MACHα1 did not exhibit self-interaction in thetwo hybrid test (FIG. 5).

Both MACHα1(C360S) and MACHβ1 coimmunoprecipitated with MORT-1 fromlysates of human embryonic kidney 293-EBNA cells, indicating that theybind to MORT-1 also in mammalian cells. Testing further if MACHα1 canbind to MORT1 also within mammalian cells, MACHα1 or MACHβ1, fused withthe FLAG octapeptide was expressed, together with HA epitope-taggedMORT1 molecules. ³⁵ [S] metabolically labeled MACHα1 and MACHβ1 fused attheir N-termini to the FLAG octapeptide (FLAG-MACHα1 and β1), and MORT1fused at its N terminus to the HA epitope (Field et al., 1988) wereexpressed in HeLa cells. Immunoprecipitation of the proteins fromlysates of the cells was performed using mouse monoclonal antibodiesagainst the FLAG octapeptide (M2; Eastman Kodak), HA epitope (12CA5,Field et al., 1988) or the p75 TNF receptor (#9, Bigda et al., 1994) asa control. The proteins were analyzed by SDS-polyacrylamide gelelectrophoresis (12% acrylamide), followed by autoradiography. BothMACHα1 and MACHβ1 co-immunoprecipitated with MORT1 from lysates of thecells, indicating that they bind to MORT1. The effectivity ofinteraction of MACHα1 with MORT1 appeared to be lower than that ofMACHβ1.

(e) MACH Molecules that Contain the CED3/ICE Homology Region can MediateCell Death:

To explore the involvement of MACH in cell-death induction, the effectof overexpression of various MACH isoforms on cell viability wasexamined. The test was performed by transfecting MACH expression vectorstogether with a β-galactosidase expression vector as a transfectionmarker into human embryonic kidney 293-EBNA cells and breast carcinomaMCF7 cells.

In brief, 293-EBNA cells, MCF7 human breast carcinoma cells and HeLaHtTA-1 cells were grown in Dulbecco's modified Eagle's minimal essentialmedium supplemented with 10% fetal calf serum, nonessential amino acids,100 U/ml penicillin and 100 μg/ml streptomycin. Cell tissue culturedishes (5×10⁵ 293-EBNA cells, 3×10⁵ MCF7 cells or 3×10⁵ HeLa cells in6-cm dishes) were transiently transfected, using the calcium phosphateprecipitation method, with the cDNAs of the indicated proteins togetherwith the β-galactosidase expression vector. In the experiments presentedin FIGS. 14A-D and 15, each dish was transfected with 3.5 μg of theindicated MACH construct and 1.5 μg of pSV-β-gal. In the experimentspresented in FIGS. 16A-D and 17-19, each dish was transfected with 2.5μg of the indicated MACH or MORT1 construct (or, as control, emptyvector) and 1.5 μg of pSV-β-gal. The cells were rinsed 6 to 10 h aftertransfection. The 293-EBNA and MCF7 cells were incubated for a further18 h without additional treatment. The HeLa cells were incubated for 26h after transfection and then for 5 h in the presence of eitheranti-Fas.APO1 antibody (CH11, 0.5 μg/ml) or TNF (100 ng/ml), togetherwith cycloheximide (10 μg/ml). The extent of cell death at the end ofthe incubation periods was assessed by determination of β-galactosidaseexpression, as described by Kumar et al., 1994.

Cultures transfected with an expression vector of either MACHα1 orMACHα2 exhibited massive cell death, manifested by cell rounding,blebbing, contraction, and finally detachment of cells from the dish(FIG. 14B). By 20 h after transfection, the majority of the transfectedcells, identified by β-galactosidase staining (X-Gal), showed condensedmorphology typical of apoptosis (FIG. 14B). In contrast, cellsexpressing the empty vector remained viable.

In particular, FIGS. 14A-D show the morphology of human embryonic kidney293-EBNA cells transiently expressing the indicated MACH isoforms. Thearrows (FIG. 14B) point to apoptopic cells. Photographs were taken 26 hafter transfection. FIG. 15 shows the quantification of MACH-induceddeath of the 293-EBNA (striped squares) and MCF7 (black squares) cellsby determination of the portion of β-galactosidase-expressing cellsexhibiting apoptopic morphology 20 h after transfection of the indicatedconstructs. Data are from three independent experiments with the293-EBNA cells and two independent experiments with the MCF7 cells. Theyare expressed as the mean percentage of the blue cells exhibiting signsof apoptosis as a fraction of the total number of blue cells counted(about 500 cells per sample).

To examine the involvement of the CED3/ICE homology region within theMACHA isoforms in their apoptopic effects, cells were transfected withthe expression vector for the MACHβ1 isoform, which lacks the CED3/ICEhomology region, as well as with expression vectors for MACHα3, whichlacks an N-terminal part of the region, and with expression vectors forMACHα1(C360S) and for a C-terminally truncated mutant of MACHα1(MACHα1(1-415)), which lacks one of the residues believed to be criticalfor CED3/ICE protease function (corresponding to Ser₃₄₇ in ICE). Nodeath (beyond the slight amount observed in cells transfected with anempty expression vector) occurred in 293-EBNA or MCF7 cells transfectedwith the expression vectors for MACHα3, MACHα1(1-415) or MACHα1(C360S).Moreover, cells transfected with MACHα1 together with these vectors alsoexhibited very little cell death, indicating that MACH molecules thatcontain an incomplete CED3/ICE region have a negative dominant effect onthe activity of the wild-type molecules. Cultures expressing MACHβ1,which does not contain the CED3/ICE region at all, did exhibit someslight cell death (FIG. 15). This effect of MACHβ1, which most probablyresults from activation of endogenous MACHα1 molecules, was for somereason more pronounced in transfected HeLa cells. Moreover, in HeLacells MACHα3, MACHα1 (1-415) and MACHα1 (C360S) were also somewhatcytotoxic (FIG. 19).

FIG. 8 diagrammatically presents the receptor and target proteininteractions participating in induction of cell death by FAS/APO1(FAS-R) and p55-R. MACHα activity appears to constitute the mostupstream enzymatic step in the cascade of signalling for the cytocidaleffects of FAS/APO1 and pS5-R. The ability of MACHβ1 to bind to bothMORT-1 and MACHα1 suggests that this isoform enhances the activity ofthe enzymatically active isoforms.

It is possible that some of the MACH isoforms serve additionalfuncitons. The ability of MACHβ1 to bind to both MORT-1 and MACHα1suggests that this isoform might enhane the acitivity of theenzymatically acitive isoforms. The mild cytoxicity observed in 293-EBNAand MCF7 cultures transfected with this isoform and the rathersignificant cytoxic effect that it exerts in HeLa cells probably reflectactivation of endogenously expressed MACHα moleucles upon binding to thetransfected MACHβ1 molecules. Conceivably, some of the MACH isoformscould also act as docking sites for molecules that are involved inother, non-cytoxic effects of Fas/APO1 and TNF receptors.

(f) Blocking of MACHα Function Interferes With Cell Death Induction byFas/APO1 and p55-R

To assess the contribution of MACHα to Fas/APO1 (FAS-R) and p55-Rcytotoxicity, MACHα3, as well as the nonfunctional MACHα1 mutants,MACHα1 (1-415) and MACHα (C360S), were expressed in cells that wereinduced to exhibit this cytotoxicity. p55-R-induced cytotoxicity wastriggered in the 293-EBNA cells by transient over-expression of thisreceptor (Boldin et al., 1995a), and Fas/APO1 cytotoxicity byover-expression of chimeric molecules comprised of the extracellulardomain of the p55-R and the transmembrane and intracellular domains ofFas/APO1. For some reason, this chimera had a far greater cytotoxiceffect than that of the normal Fas/APO1. Cytotoxic activities in HeLacells was also induced by treating them with TNF or anti-Fas/APO1antibody in the presence of the protein-synthesis blocker cycloheximide.The HeLa cells were made responsive to Fas/APO1 by transient expressionof this receptor. In all systems examined, MACHα3 and the nonfunctionalMACHα1 mutants provided effective protection against the cytotoxicityinduced by Fas/APO1 or p55-R triggering (FIGS. 16-19). Such protectionwas also observed, as previously reported (Hsu et al., 1996; Chinnaiyanet al., 1996), in cells transfected with a MORT1 N-terminal deletionmutant that lacks the MACH-binding region (MORT1(92-208)). Theseprotective effects indicate that MACHα is a necessary component of boththe Fas/APO1- and the p55-R-induced signaling cascades for cell death.

In particular, FIGS. 16A-D show morphology of 293-EBNA cells in whichcell death was induced by transient expression of a chimera comprised ofthe extracellular domain of the p55-R (amino acids 1-168) fused to thetransmembrane and intracellular domains of Fas/APO1 (amino acids153-319) (p55-Fas chimera) (FIGS. 16A and 16B), or by expression of thep55-R (FIGS. 16C and 16D), and of cells that were protected from thesecytotoxic effects by their simultaneous transfection with MACHα1 (C360S)(FIGS. 16B and 16D). Photographs were taken 26 h after transfection.FIG. 17 illustrates the quantification of death induced in 293-EBNAcells by their transfection with p55-Fas chimera or with p55-R, togetherwith an empty vector, a MORT1 deletion mutant lacking the MACH-bindingregion (MORT1(92-208)), or MACHα molecules containing a nonfunctionalCED3/ICE region. FIG. 18 shows the death of HeLa cells that transientlyexpress Fas/APO1, induced by treatment with anti-Fas/APO1 antibody(aFasY and cycloheximide (CHI), and its prevention by cotransfection ofMORT1DD(92-208), MACHα (C360S) or MACHα3. FIG. 19 shows the death ofHeLa cells induced by application of TNF and cycloheximide (CHI), andits prevention as in FIG. 18. Data are from at least two independentexperiments and are expressed as in FIGS. 14A-F and 15.

MACH is expressed in different tissues at markedly different levels andapparently also with different isotype patterns. These differencesprobably contribute to the tissue-specific features of response to theFas/APO1 ligand and TNF. As in the case of other CED3/ICE homologs (Wanget al., 1994; Alnemri et al., 1995), MACH isoforms containing incompleteCED3/ICE regions (e.g. MACHα3) are found to inhibit the activities ofcoexpressed MACHα1 or MACHα2 molecules; they are also found to blockdeath induction by Fas/APO1 and p55-R. Expression of such inhibitoryisoforms in cells may constitute a mechanism of cellular self-protectionagainst Fas/APO1- and TNF-mediated cytotoxicity. The wide heterogeneityof MACH isoforms, which greatly exceeds that observed for any of theother proteases of th CED3/ICE family, should allow a particulary finetuning of the function of the active MACH isoforms.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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34 1701 base pairs nucleic acid single linear cDNA CDS 1..768 1 GTG AATCAG GCA CCG GAG TGC AGG TTC GGG GGT GGA ATC CTT GGG CCG 48 Val Asn GlnAla Pro Glu Cys Arg Phe Gly Gly Gly Ile Leu Gly Pro 1 5 10 15 CTG GGCAAG CGG CGA GAC CTG GCC AGG GCC AGC GAG CCG AGG ACA GAG 96 Leu Gly LysArg Arg Asp Leu Ala Arg Ala Ser Glu Pro Arg Thr Glu 20 25 30 GGC GCG CGGAGG GCC GGG CCG CAG CCC CGG CCG CTT GCA GAC CCC GCC 144 Gly Ala Arg ArgAla Gly Pro Gln Pro Arg Pro Leu Ala Asp Pro Ala 35 40 45 ATG GAC CCG TTCCTG GTG CTG CTG CAC TCG GTG TCG TCC AGC CTG TCG 192 Met Asp Pro Phe LeuVal Leu Leu His Ser Val Ser Ser Ser Leu Ser 50 55 60 AGC AGC GAG CTG ACCGAG CTC AAG TTC CTA TGC CTC GGG CGC GTG GTC 240 Ser Ser Glu Leu Thr GluLeu Lys Phe Leu Cys Leu Gly Arg Val Val 65 70 75 80 AAG CGC AAG CTG GAGCGC GTG CAG AGC GGC CTA GAC CTC TTC TCC ATG 288 Lys Arg Lys Leu Glu ArgVal Gln Ser Gly Leu Asp Leu Phe Ser Met 85 90 95 CTG CTG GAG CAG AAC GACCTG GAG CCC GGG CAC ACC GAG CTC CTG CGC 336 Leu Leu Glu Gln Asn Asp LeuGlu Pro Gly His Thr Glu Leu Leu Arg 100 105 110 GAG CTG CTC GCC TCC CTGCGG CGC CAC GAC CTG CTG CGG CGC GTC GAC 384 Glu Leu Leu Ala Ser Leu ArgArg His Asp Leu Leu Arg Arg Val Asp 115 120 125 GAC TTC GAG GCG GGG GCGGCG GCC GGG GCC GCG CCT GGG GAA GAA GAC 432 Asp Phe Glu Ala Gly Ala AlaAla Gly Ala Ala Pro Gly Glu Glu Asp 130 135 140 CTG TGT GCA GCA TTT AACGTC ATA TGT GAT AAT GTG GGG AAA GAT TGG 480 Leu Cys Ala Ala Phe Asn ValIle Cys Asp Asn Val Gly Lys Asp Trp 145 150 155 160 AGA AGG CTG GCT CGTCAG CTC AAA GTC TCA GAC ACC AAG ATC GAC AGC 528 Arg Arg Leu Ala Arg GlnLeu Lys Val Ser Asp Thr Lys Ile Asp Ser 165 170 175 ATC GAG GAC AGA TACCCC CGC AAC CTG ACA GAG CGT GTG CGG GAG TCA 576 Ile Glu Asp Arg Tyr ProArg Asn Leu Thr Glu Arg Val Arg Glu Ser 180 185 190 CTG AGA ATC TGG AAGAAC ACA GAG AAG GAG AAC GCA ACA GTG GCC CAC 624 Leu Arg Ile Trp Lys AsnThr Glu Lys Glu Asn Ala Thr Val Ala His 195 200 205 CTG GTG GGG GCT CTCAGG TCC TGC CAG ATG AAC CTG GTG GCT GAC CTG 672 Leu Val Gly Ala Leu ArgSer Cys Gln Met Asn Leu Val Ala Asp Leu 210 215 220 GTA CAA GAG GTT CAGCAG GCC CGT GAC CTC CAG AAC AGG AGT GGG GCC 720 Val Gln Glu Val Gln GlnAla Arg Asp Leu Gln Asn Arg Ser Gly Ala 225 230 235 240 ATG TCC CCG ATGTCA TGG AAC TCA GAC GCA TCT ACC TCC GAA GCG TCC 768 Met Ser Pro Met SerTrp Asn Ser Asp Ala Ser Thr Ser Glu Ala Ser 245 250 255 TGATGGGCCGCTGCTTTGCG CTGGTGGACC ACAGGCATCT ACACAGCCTG GACTTTGGTT 828 CTCTCCAGGAAGGTAGCCCA GCACTGTGAA GACCCAGCAG GAAGCCAGGC TGAGTGAGCC 888 ACAGACCACCTGCTTCTGAA CTCAAGCTGC GTTTATTAAT GCCTCTCCCG CACCAGGCCG 948 GGCTTGGGCCCTGCACAGAT ATTTCCATTT CTTCCTCACT ATGACACTGA GCAAGATCTT 1008 GTCTCCACTAAATGAGCTCC TGCGGGAGTA GTTGGAAAGT TGGAACCGTG TCCAGCACAG 1068 AAGGAATCTGTGCAGATGAG CAGTCACACT GTTACTCCAC AGCGGAGGAG ACCAGCTCAG 1128 AGGCCCAGGAATCGGAGCGA AGCAGAGAGG TGGAGAACTG GGATTTGAAC CCCCGCCATC 1188 CTTCACCAGAGCCCATGCTC AACCACTGTG GCGTTCTGCT GCCCCTGCAG TTGGCAGAAA 1248 GGATGTTTTTGTCCCATTTC CTTGGAGGCC ACCGGGACAG ACCTGGACAC TAGGGTCAGG 1308 CGGGGTGCTGTGGTGGGGAG AGGCATGGCT GGGGTGGGGG TGGGGAGACC TGGTTGGCCG 1368 TGGTCCAGCTCTTGGCCCCT GTGTGAGTTG AGTCTCCTCT CTGAGACTGC TAAGTAGGGG 1428 CAGTGATGGTTGCCAGGACG AATTGAGATA ATATCTGTGA GGTGCTGATG AGTGATTGAC 1488 ACACAGCACTCTCTAAATCT TCCTTGTGAG GATTATGGGT CCTGCAATTC TACAGTTTCT 1548 TACTGTTTTGTATCAAAATC ACTATCTTTC TGATAACAGA ATTGCCAAGG CAGCGGGATC 1608 TCGTATCTTTAAAAAGCAGT CCTCTTATTC CTAAGGTAAT CCTATTAAAA CACAGCTTTA 1668 CAACTTCCATATTACAAAAA AAAAAAAAAA AAA 1701 256 amino acids amino acid linear protein2 Val Asn Gln Ala Pro Glu Cys Arg Phe Gly Gly Gly Ile Leu Gly Pro 1 5 1015 Leu Gly Lys Arg Arg Asp Leu Ala Arg Ala Ser Glu Pro Arg Thr Glu 20 2530 Gly Ala Arg Arg Ala Gly Pro Gln Pro Arg Pro Leu Ala Asp Pro Ala 35 4045 Met Asp Pro Phe Leu Val Leu Leu His Ser Val Ser Ser Ser Leu Ser 50 5560 Ser Ser Glu Leu Thr Glu Leu Lys Phe Leu Cys Leu Gly Arg Val Val 65 7075 80 Lys Arg Lys Leu Glu Arg Val Gln Ser Gly Leu Asp Leu Phe Ser Met 8590 95 Leu Leu Glu Gln Asn Asp Leu Glu Pro Gly His Thr Glu Leu Leu Arg100 105 110 Glu Leu Leu Ala Ser Leu Arg Arg His Asp Leu Leu Arg Arg ValAsp 115 120 125 Asp Phe Glu Ala Gly Ala Ala Ala Gly Ala Ala Pro Gly GluGlu Asp 130 135 140 Leu Cys Ala Ala Phe Asn Val Ile Cys Asp Asn Val GlyLys Asp Trp 145 150 155 160 Arg Arg Leu Ala Arg Gln Leu Lys Val Ser AspThr Lys Ile Asp Ser 165 170 175 Ile Glu Asp Arg Tyr Pro Arg Asn Leu ThrGlu Arg Val Arg Glu Ser 180 185 190 Leu Arg Ile Trp Lys Asn Thr Glu LysGlu Asn Ala Thr Val Ala His 195 200 205 Leu Val Gly Ala Leu Arg Ser CysGln Met Asn Leu Val Ala Asp Leu 210 215 220 Val Gln Glu Val Gln Gln AlaArg Asp Leu Gln Asn Arg Ser Gly Ala 225 230 235 240 Met Ser Pro Met SerTrp Asn Ser Asp Ala Ser Thr Ser Glu Ala Ser 245 250 255 200 base pairsnucleic acid single linear cDNA 3 CCGCCGCCGC CGCCGCCACC TGCCCAGACTTTTCTGTTCC AGGGTCAGCC TGTAGTGAAT 60 CGGCCGCTGA GCCTGAAGGA CCAACAGACGTTCGCGCGCT CTGTGGGTCT CAAATGGCGC 120 AAGGTGGGGC GCTCACTGCA GCGAGGCTGCCGGGCGCTGC GGGACCCGGC GCTGGACTCG 180 CTGGCCTACG AGTACGAGCG 200 1036 basepairs nucleic acid single linear cDNA 4 CGAGGCCACG AAGGCCGGCT GCCTGAGGAATACCAGTGGG CAAGAGAATT AGCATTTCTG 60 GAGCATCTGC TGTCTGAGCA GCCCCTGGGTGCGTCCACTT TCTGGGCACG TGAGGTTGGG 120 CCTTGGCCGC CTGAGCCCTT GAGTTGGTCACTTGAACCTT GGGAATATTG AGATTATATT 180 CTCCTGCCTT TTAAAAAGAT GGACTTCAGCAGAAATCTTT ATGATATTGG GGAACAACTG 240 GACAGTGAAG ATCTGGCCTC CCTCAAGTTCCTGAGCCTGG ACTACATTCC GCAAAGGAAG 300 CAAGAACCCA TCAAGGATGC CTTGATGTTATTCCAGAGAC TCCAGGAAAA GAGAATGTTG 360 GAGGAAAGCA ATCTGTCCTT CCTGAAGGAGCTGCTCTTCC GAATTAATAG ACTGGATTTG 420 CTGATTACCT ACCTAAACAC TAGAAAGGAGGAGATGGAAA GGGAACTTCA GACACCAGGC 480 AGGGCTCAAA TTTCTGCCTA CAGGGTCATGCTCTATCAGA TTTCAGAAGA AGTGAGCAGA 540 TCAGAATTGA GGTCTTTTAA GTTTCTTTTGCAAGAGGAAA TCTCCAAATG CAAACTGGAT 600 GATGACATGA ACCTGCTGGA TATTTTCATAGAGATGGAGA AGAGGGTCAT CCTGGGAGAA 660 GGAAAGTTGG ACATCCTGAA AAGAGTCTGTGCCCAAATCA ACAAGAGCCT GCTGAAGATA 720 ATCAACGACT ATGAAGAATT CAGCAAAGAGAGAAGCAGCA GCCTTGAAGG AAGTCCTGAT 780 GAATTTTCAA ATGACTTTGG ACAAAGTTTACCAAATGAAA AGCAAACCTC GGGGATACTG 840 TCTGATCATC AACAATCACA ATTTTGCAAAAGCACGGGAG AAAGTGCCCA AACTTCACAG 900 CATTAGGGAC AGGAATGGAA CACACTTGGATGCAGGGTTT GAGAATGTTT TTAGCTGGTG 960 GCAATAAATA TTAGAAGCCT GCAGAATCCAGCTACGAATA TAGAGGGTTT TGCTCTTGGG 1020 CCTTCGTGGC CTCGAG 1036 235 aminoacids amino acid single linear protein 5 Met Asp Phe Ser Arg Asn Leu TyrAsp Ile Gly Glu Gln Leu Asp Ser 1 5 10 15 Glu Asp Leu Ala Ser Leu LysPhe Leu Ser Leu Asp Tyr Ile Pro Gln 20 25 30 Arg Lys Gln Glu Pro Ile LysAsp Ala Leu Met Leu Phe Gln Arg Leu 35 40 45 Gln Glu Lys Arg Met Leu GluGlu Ser Asn Leu Ser Phe Leu Lys Glu 50 55 60 Leu Leu Phe Arg Ile Asn ArgLeu Asp Leu Leu Ile Thr Tyr Leu Asn 65 70 75 80 Thr Arg Lys Glu Glu MetGlu Arg Glu Leu Gln Thr Pro Gly Arg Ala 85 90 95 Gln Ile Ser Ala Tyr ArgVal Met Leu Tyr Gln Ile Ser Glu Glu Val 100 105 110 Ser Arg Ser Glu LeuArg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile 115 120 125 Ser Lys Cys LysLeu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile 130 135 140 Glu Met GluLys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu 145 150 155 160 LysArg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn 165 170 175Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Ser Ser Leu Glu Gly Ser 180 185190 Pro Asp Glu Phe Ser Asn Asp Phe Gly Gln Ser Leu Pro Asn Glu Lys 195200 205 Gln Thr Ser Gly Ile Leu Ser Asp His Gln Gln Ser Gln Phe Cys Lys210 215 220 Ser Thr Gly Glu Ser Ala Gln Thr Ser Gln His 225 230 235 78amino acids amino acid single linear protein 6 Tyr Gly Thr Leu Phe GlnAsp Leu Thr Asn Asn Ile Thr Leu Glu Asp 1 5 10 15 Leu Glu Gln Leu LysSer Ala Cys Lys Glu Asp Ile Pro Ser Glu Lys 20 25 30 Ser Glu Glu Ile ThrThr Gly Ser Ala Trp Phe Ser Phe Leu Glu Ser 35 40 45 His Asn Lys Leu AspLys Asp Asn Leu Ser Ile Ile Glu His Ile Phe 50 55 60 Glu Ile Ser Arg ArgPro Asp Leu Leu Thr Met Val Val Asp 65 70 75 479 amino acids amino acidsingle linear protein 7 Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly GluGln Leu Asp Ser 1 5 10 15 Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser LeuAsp Tyr Ile Pro Gln 20 25 30 Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu MetLeu Phe Gln Arg Leu 35 40 45 Gln Glu Lys Arg Met Leu Glu Glu Ser Asn LeuSer Phe Leu Lys Glu 50 55 60 Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu LeuIle Thr Tyr Leu Asn 65 70 75 80 Thr Arg Lys Glu Glu Met Glu Arg Glu LeuGln Thr Pro Gly Arg Ala 85 90 95 Gln Ile Ser Ala Tyr Arg Val Met Leu TyrGln Ile Ser Glu Glu Val 100 105 110 Ser Arg Ser Glu Leu Arg Ser Phe LysPhe Leu Leu Gln Glu Glu Ile 115 120 125 Ser Lys Cys Lys Leu Asp Asp AspMet Asn Leu Leu Asp Ile Phe Ile 130 135 140 Glu Met Glu Lys Arg Val IleLeu Gly Glu Gly Lys Leu Asp Ile Leu 145 150 155 160 Lys Arg Val Cys AlaGln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn 165 170 175 Asp Tyr Glu GluPhe Ser Lys Glu Arg Ser Ser Ser Leu Glu Gly Ser 180 185 190 Pro Asp GluPhe Ser Asn Gly Glu Glu Leu Cys Gly Val Met Thr Ile 195 200 205 Ser AspSer Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr Leu Asp Lys 210 215 220 ValTyr Gln Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn 225 230 235240 Asn His Asn Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His Ser 245250 255 Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr260 265 270 Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pro His Asp Asp CysThr 275 280 285 Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu MetAsp His 290 295 300 Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Leu Ser HisGly Asp Lys 305 310 315 320 Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu AlaPro Ile Tyr Glu Leu 325 330 335 Thr Ser Gln Phe Thr Gly Leu Lys Cys ProSer Leu Ala Gly Lys Pro 340 345 350 Lys Val Phe Phe Ile Gln Ala Cys GlnGly Asp Asn Tyr Gln Lys Gly 355 360 365 Ile Pro Val Glu Thr Asp Ser GluGlu Gln Pro Tyr Leu Glu Met Asp 370 375 380 Leu Ser Ser Pro Gln Thr ArgTyr Ile Pro Asp Glu Ala Asp Phe Leu 385 390 395 400 Leu Gly Met Ala ThrVal Asn Asn Cys Val Ser Tyr Arg Asn Pro Ala 405 410 415 Glu Gly Thr TrpTyr Ile Gln Ser Leu Cys Gln Ser Leu Arg Glu Arg 420 425 430 Cys Pro ArgGly Asp Asp Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr 435 440 445 Glu ValSer Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gln Met Pro 450 455 460 GlnPro Thr Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Ser Asp 465 470 475 277amino acids amino acid single linear protein 8 Met Asp Phe Ser Arg AsnLeu Tyr Asp Ile Gly Glu Gln Leu Asp Ser 1 5 10 15 Glu Asp Leu Ala SerLeu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln 20 25 30 Arg Lys Gln Glu ProIle Lys Asp Ala Leu Met Leu Phe Gln Arg Leu 35 40 45 Gln Glu Lys Arg MetLeu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu 50 55 60 Leu Leu Phe Arg IleAsn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn 65 70 75 80 Thr Arg Lys GluGlu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala 85 90 95 Gln Ile Ser AlaTyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val 100 105 110 Ser Arg SerGlu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile 115 120 125 Ser LysCys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile 130 135 140 GluMet Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu 145 150 155160 Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn 165170 175 Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Ser Ser Leu Glu Gly Ser180 185 190 Pro Asp Glu Phe Ser Asn Gly Glu Glu Leu Cys Gly Val Met ThrIle 195 200 205 Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr LeuAsp Lys 210 215 220 Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Tyr Cys LeuIle Ile Asn 225 230 235 240 Asn His Asn Phe Ala Lys Ala Arg Glu Lys ValPro Lys Leu His Ser 245 250 255 Ile Arg Asp Arg Asn Gly Thr His Leu AspAla Gly Phe Gly Asn Val 260 265 270 Phe Ser Trp Trp Gln 275 489 aminoacids amino acid single linear protein 9 Met Met Phe Ser Ser His Leu LysVal Asp Glu Ile Leu Glu Val Leu 1 5 10 15 Ile Ala Lys Gln Val Leu AsnSer Asp Asn Gly Asp Met Ile Asn Ser 20 25 30 Cys Gly Thr Val Arg Glu LysArg Arg Glu Ile Val Lys Ala Val Gln 35 40 45 Arg Arg Gly Asp Val Ala PheAsp Ala Phe Tyr Asp Ala Leu Arg Ser 50 55 60 Thr Gly His Glu Gly Leu AlaGlu Val Leu Glu Pro Leu Ala Arg Ser 65 70 75 80 Val Asp Ser Asn Ala ValGlu Phe Glu Cys Pro Met Ser Pro Ala Ser 85 90 95 His Arg Arg Ser Arg AlaLeu Ser Pro Ala Gly Tyr Thr Ser Pro Thr 100 105 110 Arg Val His Arg AspSer Val Ser Ser Val Ser Ser Phe Thr Ser Tyr 115 120 125 Gln Asp Ile TyrSer Arg Ala Arg Ser Arg Ser Arg Ser Arg Ala Leu 130 135 140 His Ser SerAsp Arg His Asn Tyr Ser Ser Pro Pro Val Asn Ala Phe 145 150 155 160 ProSer Gln Pro Ser Ser Ala Asn Ser Ser Phe Thr Gly Cys Ser Ser 165 170 175Leu Gly Tyr Ser Ser Ser Arg Asn Arg Ser Phe Ser Lys Ala Ser Gly 180 185190 Pro Thr Gln Tyr Ile Phe His Glu Glu Asp Met Asn Phe Val Asp Ala 195200 205 Pro Thr Ile Ser Arg Val Phe Asp Glu Lys Thr Met Tyr Arg Asn Phe210 215 220 Ser Ser Pro Arg Gly Met Cys Leu Ile Ile Asn Asn Glu His PheGlu 225 230 235 240 Gln Met Pro Thr Arg Asn Gly Thr Lys Ala Asp Lys AspAsn Leu Thr 245 250 255 Asn Leu Phe Arg Cys Met Gly Tyr Thr Val Ile CysLys Asp Asn Leu 260 265 270 Thr Gly Arg Gly Met Leu Leu Thr Ile Arg AspPhe Ala Lys His Glu 275 280 285 Ser His Gly Asp Ser Ala Ile Leu Val IleLeu Ser His Gly Glu Glu 290 295 300 Asn Val Ile Ile Gly Val Asp Asp IlePro Ile Ser Thr His Glu Ile 305 310 315 320 Tyr Asp Leu Leu Asn Ala AlaAsn Ala Pro Arg Leu Ala Asn Lys Pro 325 330 335 Lys Ile Val Phe Val GlnAla Cys Arg Gly Glu Arg Arg Asp Asn Gly 340 345 350 Phe Pro Val Leu AspSer Val Asp Gly Val Pro Ala Phe Leu Arg Arg 355 360 365 Gly Trp Asp AsnArg Asp Gly Pro Leu Phe Asn Phe Leu Gly Cys Val 370 375 380 Arg Pro GlnVal Gln Gln Val Trp Arg Lys Lys Pro Ser Gln Ala Asp 385 390 395 400 IleLeu Ile Arg Tyr Ala Thr Thr Ala Gln Tyr Val Ser Trp Arg Asn 405 410 415Ser Ala Arg Gly Ser Trp Phe Ile Gln Ala Val Cys Glu Val Phe Ser 420 425430 Thr His Ala Lys Asp Met Asp Val Val Glu Leu Leu Thr Glu Val Asn 435440 445 Lys Lys Val Ala Cys Gly Phe Gln Thr Ser Gln Gly Ser Asn Ile Leu450 455 460 Lys Gln Met Pro Glu Met Thr Ser Arg Leu Leu Lys Lys Phe TyrPhe 465 470 475 480 Trp Pro Glu Ala Arg Asn Ser Ala Val 485 421 aminoacids amino acid single linear protein 10 Met His Pro His His Gln GluThr Leu Lys Lys Asn Arg Val Val Leu 1 5 10 15 Ala Lys Gln Leu Leu LeuSer Glu Leu Leu Glu His Leu Leu Glu Lys 20 25 30 Asp Ile Ile Thr Leu GluMet Arg Glu Leu Ile Gln Ala Lys Val Gly 35 40 45 Ser Phe Ser Gln Asn ValGlu Leu Leu Asn Leu Leu Pro Lys Arg Gly 50 55 60 Pro Gln Ala Phe Asp AlaPhe Cys Glu Ala Leu Arg Glu Thr Lys Gln 65 70 75 80 Gly His Leu Glu AspMet Leu Leu Thr Thr Leu Ser Gly Leu Gln His 85 90 95 Val Leu Pro Pro LeuSer Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro 100 105 110 Val Cys Glu SerCys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp 115 120 125 Thr Val GluHis Ser Leu Asp Asn Lys Asp Gly Pro Val Cys Leu Gln 130 135 140 Val LysPro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala 145 150 155 160Tyr Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn 165 170175 Val His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp 180185 190 Val Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp195 200 205 Val His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu LysLeu 210 215 220 Gln Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp SerCys Ile 225 230 235 240 Val Ala Leu Leu Ser His Gly Val Glu Gly Ala IleTyr Gly Val Asp 245 250 255 Gly Lys Leu Leu Gln Leu Gln Glu Val Phe GlnLeu Phe Asp Asn Ala 260 265 270 Asn Cys Pro Ser Leu Gln Asn Lys Pro LysMet Phe Phe Ile Gln Ala 275 280 285 Cys Arg Gly Asp Glu Thr Asp Arg GlyVal Asp Gln Gln Asp Gly Lys 290 295 300 Asn His Ala Gly Ser Pro Gly CysGlu Glu Ser Asp Ala Gly Lys Glu 305 310 315 320 Lys Leu Pro Lys Met ArgLeu Pro Thr Arg Ser Asp Met Ile Cys Gly 325 330 335 Tyr Ala Cys Leu LysGly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly 340 345 350 Ser Trp Tyr IleGlu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys 355 360 365 Asp Met HisVal Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys 370 375 380 Asp ArgGlu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu 385 390 395 400Met Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro 405 410415 Gly His Pro Pro Thr 420 376 amino acids amino acid single linearprotein 11 Val Lys Lys Asp Asn His Lys Lys Lys Thr Val Lys Met Leu GluTyr 1 5 10 15 Leu Gly Lys Asp Val Leu His Gly Val Phe Asn Tyr Leu AlaLys His 20 25 30 Asp Val Leu Thr Leu Lys Glu Glu Glu Lys Lys Lys Tyr TyrAsp Ala 35 40 45 Lys Ile Glu Asp Lys Ala Leu Ile Leu Val Asp Ser Leu ArgLys Asn 50 55 60 Arg Val Ala His Gln Met Phe Thr Gln Thr Leu Leu Asn MetAsp Gln 65 70 75 80 Lys Ile Thr Ser Val Lys Pro Leu Leu Gln Ile Glu AlaGly Pro Pro 85 90 95 Glu Ser Ala Glu Ser Thr Asn Ile Leu Lys Leu Cys ProArg Glu Glu 100 105 110 Phe Leu Arg Leu Cys Lys Lys Asn His Asp Glu IleTyr Pro Ile Lys 115 120 125 Lys Arg Glu Asp Arg Arg Arg Leu Ala Leu IleIle Cys Asn Thr Lys 130 135 140 Phe Asp His Leu Pro Ala Arg Asn Gly AlaHis Tyr Asp Ile Val Gly 145 150 155 160 Met Lys Arg Leu Leu Gln Gly LeuGly Tyr Thr Val Val Asp Glu Lys 165 170 175 Asn Leu Thr Ala Arg Asp MetGlu Ser Val Leu Arg Ala Phe Ala Ala 180 185 190 Arg Pro Glu His Lys SerSer Asp Ser Thr Phe Leu Val Leu Met Ser 195 200 205 His Gly Ile Leu GluGly Ile Cys Gly Thr Ala His Lys Lys Lys Lys 210 215 220 Pro Asp Val LeuLeu Tyr Asp Thr Ile Phe Gln Ile Phe Asn Asn Arg 225 230 235 240 Asn CysLeu Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Val Gln Ala 245 250 255 CysArg Gly Glu Lys His Gly Glu Leu Trp Val Arg Asp Ser Pro Ala 260 265 270Ser Leu Ala Val Ile Ser Ser Gln Ser Ser Glu Asn Leu Glu Ala Asp 275 280285 Ser Val Cys Lys Ile His Glu Glu Lys Asp Phe Ile Ala Phe Cys Ser 290295 300 Ser Thr Pro His Asn Val Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile305 310 315 320 Phe Ile Thr Glu Leu Ile Thr Cys Phe Gln Lys Tyr Ser CysCys Cys 325 330 335 His Leu Met Glu Ile Phe Arg Lys Val Gln Lys Ser PheGlu Val Pro 340 345 350 Gln Ala Lys Ala Gln Met Pro Thr Ile Glu Arg AlaThr Leu Thr Arg 355 360 365 Asp Phe Tyr Leu Phe Pro Gly Asn 370 375 377amino acids amino acid single linear protein 12 Met Ala Glu Gly Asn HisArg Lys Lys Pro Leu Lys Val Leu Glu Ser 1 5 10 15 Leu Gly Lys Asp PheLeu Thr Gly Val Leu Asp Asn Leu Val Glu Gln 20 25 30 Asn Val Leu Asn TrpLys Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala 35 40 45 Lys Thr Glu Asp LysVal Arg Val Met Ala Asp Ser Met Gln Glu Lys 50 55 60 Gln Arg Met Ala GlyGln Met Leu Leu Gln Thr Phe Phe Asn Ile Asp 65 70 75 80 Gln Ile Ser ProAsn Lys Lys Ala His Pro Asn Met Glu Ala Gly Pro 85 90 95 Pro Glu Ser GlyGlu Ser Thr Asp Ala Leu Lys Leu Cys Pro His Glu 100 105 110 Glu Phe LeuArg Leu Cys Lys Glu Arg Ala Glu Glu Ile Tyr Pro Ile 115 120 125 Lys GluArg Asn Asn Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Thr 130 135 140 GluPhe Asp His Leu Pro Pro Arg Asn Gly Ala Asp Phe Asp Ile Thr 145 150 155160 Gly Met Lys Glu Leu Leu Glu Gly Leu Asp Tyr Ser Val Asp Val Glu 165170 175 Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Ala Leu Arg Ala Phe Ala180 185 190 Thr Arg Pro Glu His Lys Ser Ser Asp Ser Thr Phe Leu Val LeuMet 195 200 205 Ser His Gly Ile Leu Glu Gly Ile Cys Gly Thr Val His AspGlu Lys 210 215 220 Lys Pro Asp Val Leu Leu Tyr Asp Thr Ile Phe Gln IlePhe Asn Asn 225 230 235 240 Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro LysVal Ile Ile Val Gln 245 250 255 Ala Cys Arg Gly Ala Asn Arg Gly Glu LeuTrp Val Arg Asp Ser Pro 260 265 270 Ala Ser Leu Glu Val Ala Ser Ser GlnSer Ser Glu Asn Leu Glu Glu 275 280 285 Asp Ala Val Tyr Lys Thr His ValGlu Lys Asp Phe Ile Ala Phe Cys 290 295 300 Ser Ser Thr Pro His Asn ValSer Trp Arg Asp Ser Thr Met Gly Ser 305 310 315 320 Ile Phe Ile Thr GlnLeu Ile Thr Cys Phe Gln Lys Tyr Ser Trp Cys 325 330 335 Cys His Leu GluGlu Val Phe Arg Lys Val Gln Gln Ser Phe Glu Thr 340 345 350 Pro Arg AlaLys Ala Gln Met Pro Thr Ile Glu Arg Leu Ser Met Thr 355 360 365 Arg TyrPhe Tyr Leu Phe Pro Gly Asn 370 375 404 amino acids amino acid singlelinear protein 13 Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu PheIle Arg Ser 1 5 10 15 Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp GluLeu Leu Gln Thr 20 25 30 Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val LysArg Glu Asn Ala 35 40 45 Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp SerVal Ile Pro Lys 50 55 60 Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr IleCys Glu Glu Asp 65 70 75 80 Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser AlaAsp Gln Thr Ser Gly 85 90 95 Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly ValLeu Ser Ser Phe Pro 100 105 110 Ala Pro Gln Ala Val Gln Asp Asn Pro AlaMet Pro Thr Ser Ser Gly 115 120 125 Ser Glu Gly Asn Val Lys Leu Cys SerLeu Glu Glu Ala Gln Arg Ile 130 135 140 Trp Lys Gln Lys Ser Ala Glu IleTyr Pro Ile Met Asp Lys Ser Ser 145 150 155 160 Arg Thr Arg Leu Ala LeuIle Ile Cys Asn Glu Glu Phe Asp Ser Ile 165 170 175 Pro Arg Arg Thr GlyAla Glu Val Asp Ile Thr Gly Met Thr Met Leu 180 185 190 Leu Gln Asn LeuGly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala 195 200 205 Ser Asp MetThr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His 210 215 220 Lys ThrSer Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg 225 230 235 240Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu 245 250255 Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser 260265 270 Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp275 280 285 Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser GlyAsn 290 295 300 Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala IleLys Lys 305 310 315 320 Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys SerSer Thr Pro Asp 325 330 335 Asn Val Ser Trp Arg His Pro Thr Met Gly SerVal Phe Ile Gly Arg 340 345 350 Leu Ile Glu His Met Gln Glu Tyr Ala CysSer Cys Asp Val Glu Glu 355 360 365 Ile Phe Arg Lys Val Arg Phe Ser PheGlu Gln Pro Asp Gly Arg Ala 370 375 380 Gln Met Pro Thr Thr Glu Arg ValThr Leu Thr Arg Cys Phe Tyr Leu 385 390 395 400 Phe Pro Gly His 2887base pairs nucleic acid single linear cDNA 14 GATTCTGCCT TTCTGCTGGAGGGAAGTGTT TTCACAGGTT CTCCTCCTTT TATCTTTTGT 60 GTTTTTTTTC AAGCCCTGCTGAATTTGCTA GTCAACTCAA CAGGAAGTGA GGCCATGGAG 120 GGAGGCAGAA GAGCCAGGGTGGTTATTGAA AGTAGAAGAA ACTTCTTCCT GGGAGCCTTT 180 CCCACCCCCT TCCCTGCTGAGCACGTGGAG TTAGGCAGGT TAGGGGACTC GGAGACTGCG 240 ATGGTGCCAG GAAAGGGTGGAGCGGATTAT ATTCTCCTGC CTTTTAAAAA GATGGACTTC 300 AGCAGAAATC TTTATGATATTGGGGAACAA CTGGACAGTG AAGATCTGGC CTCCCTCAAG 360 TTCCTGAGCC TGGACTACATTCCGCAAAGG AAGCAAGAAC CCATCAAGGA TGCCTTGATG 420 TTATTCCAGA GACTCCAGGAAAAGAGAATG TTGGAGGAAA GCAATCTGTC CTTCCTGAAG 480 GAGCTGCTCT TCCGAATTAATAGACTGGAT TTGCTGATTA CCTACCTAAA CACTAGAAAG 540 GAGGAGATGG AAAGGGAACTTCAGACACCA GGCAGGGCTC AAATTTCTGC CTACAGGGTC 600 ATGCTCTATC AGATTTCAGAAGAAGTGAGC AGATCAGAAT TGAGGTCTTT TAAGTTTCTT 660 TTGCAAGAGG AAATCTCCAAATGCAAACTG GATGATGACA TGAACCTGCT GGATATTTTC 720 ATAGAGATGG AGAAGAGGGTCATCCTGGGA GAAGGAAAGT TGGACATCCT GAAAAGAGTC 780 TGTGCCCAAA TCAACAAGAGCCTGCTGAAG ATAATCAACG ACTATGAAGA ATTCAGCAAA 840 GAGAGAAGCA GCAGCCTTGAAGGAAGTCCT GATGAATTTT CAAATGGGGA GGAGTTGTGT 900 GGGGTAATGA CAATCTCGGACTCTCCAAGA GAACAGGATA GTGAATCACA GACTTTGGAC 960 AAAGTTTACC AAATGAAAAGCAAACCTCGG GGATACTGTC TGATCATCAA CAATCACAAT 1020 TTTGCAAAAG CACGGGAGAAAGTGCCCAAA CTTCACAGCA TTAGGGACAG GAATGGAACA 1080 CACTTGGATG CAGGGGCTTTGACCACGACC TTTGAAGAGC TTCATTTTGA GATCAAGCCC 1140 CACGATGACT GCACAGTAGAGCAAATCTAT GAGATTTTGA AAATCTACCA ACTCATGGAC 1200 CACAGTAACA TGGACTGCTTCATCTGCTGT ATCCTCTCCC ATGGAGACAA AGGCATCATC 1260 TATGGCACTG ATGGACAGGAGGCCCCCATC TATGAGCTGA CATCTCAGTT CACTGGTTTG 1320 AAGTGCCCTT CCCTTGCTGGAAAACCCAAA GTGTTTTTTA TTCAGGCTTG TCAGGGGGAT 1380 AACTACCAGA AAGGTATACCTGTTGAGACT GATTCAGAGG AGCAACCCTA TTTAGAAATG 1440 GATTTATCAT CACCTCAAACGAGATATATC CCGGATGAGG CTGACTTTCT GCTGGGGATG 1500 GCCACTGTGA ATAACTGTGTTTCCTACCGA AACCCTGCAG AGGGAACCTG GTACATCCAG 1560 TCACTTTGCC AGAGCCTGAGAGAGCGATGT CCTCGAGGCG ATGATATTCT CACCATCCTG 1620 ACTGAAGTGA ACTATGAAGTAAGCAACAAG GATGACAAGA AAAACATGGG GAAACAGATG 1680 CCTCAGCCTA CTTTCACACTAAGAAAAAAA CTTGTCTTCC CTTCTGATTG ATGGTGCTAT 1740 TTTGTTTGTT TTGTTTTGTTTTGTTTTTTT GAGACAGAAT CTCGCTCTGT CGCCCAGGCT 1800 GGAGTGCAGT GGCGTGATCTCGGCTCACCG CAAGCTCCGC CTCCCGGGTT CACGCCATTC 1860 TCCTGCCTCA GCCTCCCGAGTAGCTGGGAC TACAGGGGCC CGCCACCACA CCTGGCTAAT 1920 TTTTTAAAAA TATTTTTAGTAGAGACAGGG TTTCACTGTG TTAGCCAGGG TGGTCTTGAT 1980 CTCCTGACCT CGTGATCCACCCACCTCGGC CTCCCAAAGT GCTGGGATTA CAGGCGTGAG 2040 CCACCGCGCC TGGCCGATGGTACTATTTAG ATATAACACT ATGTTTATTT ACTAATTTTC 2100 TAGATTTTCT ACTTTATTAATTGTTTTGCA CTTTTTTATA AGAGCTAAAG TTAAATAGGA 2160 TATTAACAAC AATAACACTGTCTCCTTTCT CTTACGCTTA AGGCTTTGGG AATGTTTTTA 2220 GCTGGTGGCA ATAAATACCAGACACGTACA AAATCCAGCT ATGAATATAG AGGGCTTATG 2280 ATTCAGATTG TTATCTATCAACTATAAGCC CACTGTTAAT ATTCTATTAA CTTTAATTCT 2340 CTTTCAAAGC TAAATTCCACACTACCACAT TAAAAAAATT AGAAAGTAGC CACGTATGGT 2400 GGCTCATGTC TATAATCCCAGCACTTTGGG AGGTTGAGGT GGGAGGATTT GCTTGAACCC 2460 AAGAGGTCCA AGGCTGCAGTGAGCCATGTT CACACCGCTG CACTCAAGCT TGGGTGACAG 2520 AGCAAGACCC CGTCCCCAAAAAAATTTTTT TTTTAATAAA CCCAAATTTG TTTGAAAACT 2580 TTTAAAAATT CAAATGATTTTTACAAGTTT TAAATAAGCT CTCCCCAAAC TTGCTTTATG 2640 CCTTCTTATT GCTTTTATGATATATATATG CTTGGCTAAC TATATTTGCT TTTTGCTAAC 2700 AATGCTCTGG GGTCTTTTTATGCATTTGCA TTTGCTCTTT CATCTCTGCT TGGATTATTT 2760 TAAATCATTA GGAATTAAGTTATCTTTAAA ATTTAAGTAT CTTTTTTCCA AAACATTTTT 2820 TAATAGAATA AAATATAATTTGATCTTAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2880 AAAAAAA 2887 1323 basepairs nucleic acid single linear cDNA 15 AAATGCAAAC TGGATGATGACATGAACCTG CTGGATATTT TCATAGAGAT CGAGAAGAGG 60 GTCATCCTGG GAGAAGGAAAGTTGGACATC CTGAAAAGAG TCTGTGCCCA AATCAACAAG 120 AGCCTGCTGA AGATAATCAACGACTATGAA GAATTCAGCA AAGGGGAGGA GTTGTGTGGG 180 GTAATGACAA TCTCGGACTCTCCAAGAGAA CAGGATAGTG AATCACAGAC TTTGGACAAA 240 GTTTACCAAA TGAAAAGCAAACCTCGGGGA TACTGTCTGA TCATCAACAA TCACAATTTT 300 GCAAAAGCAC GGGAGAAAGTGCCCAAACTT CACAGCATTA GGGACAGGAA TGGAACACAC 360 TTGGATGCAG GGGCTTTGACCACGACCTTT GAAGAGCTTC ATTTTGAGAT CAAGCCCCAC 420 GATGACTGCA CAGTAGAGCAAATCTATGAG ATTTTGAAAA TCTACCAACT CATGGACCAC 480 AGTAACATGG ACTGCTTCATCTGCTGTATC CTCTCCCATG GAGACAAAGG CATCATCTAT 540 GGCACTGATG GACAGGAGGCCCCCATCTAT GAGCTGACAT CTCAGTTCAC TGGTTTGAAG 600 TGCCCTTCCC TTGCTGGAAAACCCAAAGTG TTTTTTATTC TTATCATCAC CTCAAACGAG 660 ATATATCCCG GATGAGGCTGACTTTCTGCT GGGGATGGCC ACTGTGAATA ACTGTGTTTC 720 CTACCGAAAC CCTGCAGAGGGAACCTGGTA CATCCAGTCA CTTTGCCAGA GCCTGAGAGA 780 GCGATGTCCT CGAGGCGATGATATTCTCAC CATCCTGACT GAAGTGAACT ATGAAGTAAG 840 CAACAAGGAT GACAAGAAAAACATGGGGAA ACAGATGCCT CAGCCTACTT TCACACTAAG 900 AAAAAAACTT GTCTTCCCTTCTGATTGATG GTGCTATTTT GTTTGTTTTG TTTTGTTTTG 960 TTTTTTTGAG ACAGAATCTCGCTCTGTCGC CCAGGCTGGA GTGCAGTGGC GTGATCTCGG 1020 CTCACCGCAA GCTCCGCCTCCCGGGTTCAC GCCATTCTCC TGCCTCAGCC TCCCGAGTAG 1080 CTGGGACTAC AGGGGCCCGCCACCACACCT GGCTAATTTT TTAAAAATAT TTTTAGTAGA 1140 GACAGGGTTT CACTGTGTTAGCCAGGGTGG TCTTGATCTC CTGACCTCGT GATCCACCCA 1200 CCTCGGCCTC CCAAAGTGCTGGGATTACAG GCGTGAGCCA CCGCGCCTGG CCGATGGTAC 1260 TATTTAGATA TAACACTATGTTTATTTACT AATTTTCTAG ATTTTCTACT TTATTAATTG 1320 TTT 1323 335 aminoacids amino acid single linear protein 16 Lys Cys Lys Leu Asp Asp AspMet Asn Leu Leu Asp Ile Phe Ile Glu 1 5 10 15 Met Glu Lys Arg Val IleLeu Gly Glu Gly Lys Leu Asp Ile Leu Lys 20 25 30 Arg Val Cys Ala Gln IleAsn Lys Ser Leu Leu Lys Ile Ile Asn Asp 35 40 45 Tyr Glu Glu Phe Ser LysGly Glu Glu Leu Cys Gly Val Met Thr Ile 50 55 60 Ser Asp Ser Pro Arg GluGln Asp Ser Glu Ser Gln Thr Leu Asp Lys 65 70 75 80 Val Tyr Gln Met LysSer Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn 85 90 95 Asn His Asn Phe AlaLys Ala Arg Glu Lys Val Pro Lys Leu His Ser 100 105 110 Ile Arg Asp ArgAsn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr 115 120 125 Thr Phe GluGlu Leu His Phe Glu Ile Lys Pro His Asp Asp Cys Thr 130 135 140 Val GluGln Ile Tyr Glu Ile Trp Lys Ile Tyr Gln Leu Met Asp His 145 150 155 160Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gly Asp Lys 165 170175 Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Gly Pro Ile Tyr Glu Leu 180185 190 Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro195 200 205 Lys Val Phe Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln LysGly 210 215 220 Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu GluMet Asp 225 230 235 240 Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp GluAla Asp Phe Leu 245 250 255 Leu Gly Met Ala Thr Val Asn Asn Cys Val SerTyr Arg Asn Pro Ala 260 265 270 Glu Gly Thr Trp Tyr Ile Gln Ser Leu CysGln Ser Leu Arg Glu Arg 275 280 285 Cys Pro Arg Gly Asp Asp Ile Leu ThrIle Leu Thr Glu Val Asn Tyr 290 295 300 Glu Val Ser Asn Lys Asp Asp LysLys Asn Met Gly Lys Gln Met Pro 305 310 315 320 Gln Pro Thr Phe Thr LeuArg Lys Lys Leu Val Phe Pro Ser Asp 325 330 335 2619 base pairs nucleicacid single linear cDNA 17 GTAGTGGATA GGCCTGTGAC GAAGGTGCTA CCATCGTGAGAGTAAGATTA TATTCTCCTG 60 CCTTTTAAAA AGATGGACTT CAGCAGAAAT CTTTATGATATTGGGGAACA ACTGGACAGT 120 GAAGATCTGG CCTCCCTCAA GTTCCTGAGC CTGGACTACATTCCGCAAAG GAAGCAAGAA 180 CCCATCAAGG ATGCCTTGAT GTTATTCCAG AGACTCCAGGAAAAGAGAAT GTTGGAGGAA 240 AGCAATCTGT CCTTCCTGAA GGAGCTGCTC TTCCGAATTAATAGACTGGA TTTGCTGATT 300 ACCTACCTAA ACACTAGAAA GGAGGAGATG GAAAGGGAACTTCAGACACC AGGCAGGGCT 360 CAAATTTCTG CCTACAGGGT CATGCTCTAT CAGATTTCAGAAGAAGTGAG CAGATCAGAA 420 TTGAGGTCTT TTAAGTTTCT TTTGCAAGAG GAAATCTCCAAATGCAAACT GGATGATGAC 480 ATGAACCTGC TGGATATTTT CATAGAGATG GAGAAGAGGGTCATCCTGGG AGAAGGAAAG 540 TTGGACATCC TGAAAAGAGT CTGTGCCCAA ATCAACAAGAGCCTGCTGAA GATAATCAAC 600 GACTATGAAG AATTCAGCAA AGGGGAGGAG TTGTGTGGGGTAATGACAAT CTCGGACTCT 660 CCAAGAGAAC AGGATAGTGA ATCACAGACT TTGGACAAAGTTTACCAAAT GAAAAGCAAA 720 CCTCGGGGAT ACTGTCTGAT CATCAACAAT CACAATTTTGCAAAAGCACG GGAGAAAGTG 780 CCCAAACTTC ACAGCATTAG GGACAGGAAT GGAACACACTTGGATGCAGG GGCTTTGACC 840 ACGACCTTTG AAGAGCTTCA TTTTGAGATC AAGCCCCACGATGACTGCAC AGTAGAGCAA 900 ATCTATGAGA TTTTGAAAAT CTACCAACTC ATGGACCACAGTAACATGGA CTGCTTCATC 960 TGCTGTATCC TCTCCCATGG AGACAAAGGC ATCATCTATGGCACTGATGG ACAGGAGGCC 1020 CCCATCTATG AGCTGACATC TCAGTTCACT GGTTTGAAGTGCCCTTCCCT TGCTGGAAAA 1080 CCCAAAGTGT TTTTTATTCA GGCTTGTCAG GGGGATAACTACCAGAAAGG TATACCTGTT 1140 GAGACTGATT CAGAGGAGCA ACCCTATTTA GAAATGGATTTATCATCACC TCAAACGAGA 1200 TATATCCCGG ATGAGGCTGA CTTTCTGCTG GGGATGGCCACTGTGAATAA CTGTGTTTCC 1260 TACCGAAACC CTGCAGAGGG AACCTGGTAC ATCCAGTCACTTTGCCAGAG CCTGAGAGAG 1320 CGATGTCCTC GAGGCGATGA TATTCTCACC ATCCTGACTGAAGTGAACTA TGAAGTAAGC 1380 AACAAGGATG ACAAGAAAAA CATGGGGAAA CAGATGCCTCAGCCTACTTT CACACTAAGA 1440 AAAAAACTTG TCTTCCCTTC TGATTGATGG TGCTATTTTGTTTGTTTTGT TTTGTTTTGT 1500 TTTTTTGAGA CAGAATCTCG CTCTGTCGCC CAGGCTGGAGTGCAGTGGCG TGATCTCGGC 1560 TCACCGCAAG CTCCGCCTCC CGGGTTCACG CCATTCTCCTGCCTCAGCCT CCCGAGTAGC 1620 TGGGACTACA GGGGCCCGCC ACCACACCTG GCTAATTTTTTAAAAATATT TTTAGTAGAG 1680 ACAGGGTTTC ACTGTGTTAG CCAGGGTGGT CTTGATCTCCTGACCTCGTG ATCCACCCAC 1740 CTCGGCCTCC CAAAGTGCTG GGATTACAGG CGTGAGCCACCGCGCCTGGC CGATGGTACT 1800 ATTTAGATAT AACACTATGT TTATTTACTA ATTTTCTAGATTTTCTACTT TATTAATTGT 1860 TTTGCACTTT TTTATAAGAG CTAAAGTTAA ATAGGATATTAACAACAATA ACACTGTCTC 1920 CTTTCTCTTA TGCTTAAGGC TTTGGGAATG TTTTTAGCTGGTGGCAATAA ATACCAGACA 1980 CGTACAAAAT CCAGCTATGA ATATAGAGGG CTTATGATTCAGATTGTTAT CTATCAACTA 2040 TAAGCCCACT GTTAATATTC TATTAACTTT AATTCTCTTTCAAAGCTAAA TTCCACACTA 2100 CCACATTAAA AAAATTAGAA AGTAGCCACG TATGGTGGCTCATGTCTATA ATCCCAGCAC 2160 TTTGGGAGGT TGAGGTGGGA GGATTTGCTT GAACCCAAGAGGTCCAAGGC TGCAGTGAGC 2220 CATGTTCACA CCGCTGCACT CAAGCTTGGG TGACAGAGCAAGACCCCGTC CCCAAAAAAA 2280 TTTTTTTTTT AATAAACCCA AATTTGTTTG AAAACTTTTAAAAATTCAAA TGATTTTTAC 2340 AAGTTTTAAA TAAGCTCTCC CCAAACTTGC TTTATGCCTTCTTATTGCTT TTATGATATA 2400 TATATGCTTG GCTAACTATA TTTGCTTTTT GCTAACAATGCTCTGGGGTC TTTTTATGCA 2460 TTTGCATTTG CTCTTTCATC TCTGCTTGGA TTATTTTAAATCATTAGGAA TTAAGTTATC 2520 TTTAAAATTT AAGTATCTTT TTTCCAAAAC ATTTTTTAATAGAATAAAAT ATAATTTGAT 2580 CTTAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAA2619 464 amino acids amino acid single linear peptide 18 Met Asp Phe SerArg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser 1 5 10 15 Glu Asp LeuAla Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln 20 25 30 Arg Lys GlnGlu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu 35 40 45 Gln Glu LysArg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu 50 55 60 Leu Leu PheArg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn 65 70 75 80 Thr ArgLys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala 85 90 95 Gln IleSer Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val 100 105 110 SerArg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile 115 120 125Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile 130 135140 Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu 145150 155 160 Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile IleAsn 165 170 175 Asp Tyr Glu Glu Phe Ser Lys Gly Glu Glu Leu Cys Gly ValMet Thr 180 185 190 Ile Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser GlnThr Leu Asp 195 200 205 Lys Val Tyr Gln Met Lys Ser Lys Pro Arg Gly TyrCys Leu Ile Ile 210 215 220 Asn Asn His Asn Phe Ala Lys Ala Arg Glu LysVal Pro Lys Leu His 225 230 235 240 Ser Ile Arg Asp Arg Asn Gly Thr HisLeu Asp Ala Gly Ala Leu Thr 245 250 255 Thr Thr Phe Glu Glu Leu His PheGlu Ile Lys Pro His Asp Asp Cys 260 265 270 Thr Val Glu Gln Ile Tyr GluIle Leu Lys Ile Tyr Gln Leu Met Asp 275 280 285 His Ser Asn Met Asp CysPhe Ile Cys Cys Ile Leu Ser His Gly Asp 290 295 300 Lys Gly Ile Ile TyrGly Thr Asp Gly Gln Glu Ala Pro Ile Tyr Glu 305 310 315 320 Leu Thr SerGln Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gly Lys 325 330 335 Pro LysVal Phe Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys 340 345 350 GlyIle Pro Val Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met 355 360 365Asp Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe 370 375380 Leu Leu Gly Met Ala Thr Val Asn Asn Cys Val Ser Tyr Arg Asn Pro 385390 395 400 Ala Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu ArgGlu 405 410 415 Arg Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Leu Thr GluVal Asn 420 425 430 Tyr Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Met GlyLys Gln Met 435 440 445 Pro Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu ValPhe Pro Ser Asp 450 455 460 1301 base pairs nucleic acid single linearcDNA 19 CCAAATGCAA ACTGGATGAT GACATGAACC TGCTGGATAT TTTCATAGAGATGGAGAAGAA 60 GGGTCATCCT GGGAGAAGGA AAGTTGGACA TCCTGAAAAG AGTCTGTGCCCAAATCAACA 120 AGAGCCTGCT GAAGATAATC AACGACTATG AAGAATTCAG CAAAGGGGCTTTGACCACGA 180 CCTTTGAAGA GCTTCATTTT GAGATCAAGC CCCACGATGA CTGCACAGTAGAGCAAATCT 240 ATGAGATTTT GAAAATCTAC CAACTCATGG ACCACAGTAA CATGGACTGCTTCATCTGCT 300 GTATCCTCTC CCATGGAGAC AAAGGCATCA TCTATGGCAC TGATGGACAGGAGGCCCCCA 360 TCTATGAGCT GACATCTCAG TTCACTGGTT TGAAGTGCCC TTCCCTTGCTGGAAAACCCA 420 AAGTGTTTTT TATTCAGGCT TGTCAGGGGG ATAACTACCA GAAAGGTATACCTGTTGAGA 480 CTGATTCAGA GGAGCAACCC TATTTAGAAA TGGATTTATC ATCACCTCAAACGAGATATA 540 TCCCGGATGA GGCTGACTTT CTGCTGGGGA TGGCCACTGT GAATAACTGTGTTTCCTACC 600 GAAACCCTGC AGAGGGAACC TGGTACATCC AGTCACTTTG CCAGAGCCTGAGAGAGCGAT 660 GTCCTCGAGG CGATGATATT CTCACCATCC TGACTGAAGT GAACTATGAAGTAAGCAACA 720 AGGATGACAA GAAAAACATG GGGAAACAGA TGCCTCAGCC TACTTTCACACTAAGAAAAA 780 AACTTGTCTT CCCTTCTGAT TGATGGTGCT ATTTTGTTTG TTTTGTTTTGTTTTGTTTTT 840 TTGAGACAGA ATCTCGCTCT GTCGCCCAGG CTGGAGTGCA GTGGCGTGATCTCGGCTCAC 900 CGCGAGCTCC GCCTCCCGGG TTCACGCCAT TCTCCTGCCT CAGCCTCCCGAGTAGCTGGG 960 ACTACAGGGG CCCGCCATCA CACCTGGCTA ATTTTTTAAA AATATTTTTAGTAGAGACAG 1020 GGTTTCACTG TGTTAGCCAG GGTGGTCTTG ATCTCCTGAC CTCGTGATCCACCCACCTCG 1080 GCCTCCCAAA GTGCTGGGAT TACAGGCGTG AGCCACCGCG CCTGGCCGATGGTACTATTT 1140 AGATATAACA CTATGTTTAT TTACTAATTT TCTAGATTTT CTACTTTATTAATTGTTTTG 1200 CACTTTTTTA TAAGAGCTAA AGTTAAATAG GATATTAACA ACAATAACACTGTCTCCTTT 1260 CTCTTACGCT TAAGGCTTTG GGAATGTTTT TAGCTGGTGG C 1301 266amino acids amino acid single linear protein 20 Lys Cys Lys Leu Asp AspAsp Met Asn Leu Leu Asp Ile Phe Ile Glu 1 5 10 15 Met Glu Lys Arg ValIle Leu Gly Glu Gly Lys Leu Asp Ile Leu Lys 20 25 30 Arg Val Cys Ala GlnIle Asn Lys Ser Leu Leu Lys Ile Ile Asn Asp 35 40 45 Tyr Glu Glu Phe SerLys Gly Ala Leu Thr Thr Thr Phe Glu Glu Leu 50 55 60 His Phe Glu Ile LysPro His Asp Asp Cys Thr Val Glu Gln Ile Tyr 65 70 75 80 Glu Ile Leu LysIle Tyr Gln Leu Met Asp His Ser Asn Met Asp Cys 85 90 95 Phe Ile Cys CysIle Leu Ser His Gly Asp Lys Gly Ile Ile Tyr Gly 100 105 110 Thr Asp GlyGln Glu Ala Pro Ile Tyr Glu Leu Thr Ser Gln Phe Thr 115 120 125 Gly LeuLys Cys Pro Ser Leu Ala Gly Lys Pro Lys Val Phe Phe Ile 130 135 140 GlnAla Cys Gln Gly Asp Asn Tyr Gln Lys Gly Ile Pro Val Glu Thr 145 150 155160 Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met Asp Leu Ser Ser Pro Gln 165170 175 Thr Arg Tyr Ile Pro Asp Glu Ala Asp Phe Leu Leu Gly Met Ala Thr180 185 190 Val Asn Asn Cys Val Ser Tyr Arg Asn Pro Ala Glu Gly Thr TrpTyr 195 200 205 Ile Gln Ser Leu Cys Gln Ser Leu Arg Glu Arg Cys Pro ArgGly Asp 210 215 220 Asp Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr Glu ValSer Asn Lys 225 230 235 240 Asp Asp Lys Lys Asn Met Gly Lys Gln Met ProGln Pro Thr Phe Thr 245 250 255 Leu Arg Lys Lys Leu Val Phe Pro Ser Asp260 265 334 base pairs nucleic acid single linear cDNA 21 CCAAATGCAAACTGGATGAT GACATGAACC TGCTGGATAT TTTCATAGAG ATGGAGAAGA 60 GGGTCATCCTGGGAGAAGGA AAGTTGGACA TCCTGAAAAG AGTCTGTGCC CAAATCAACA 120 AGAGCCTGCTGAAGATAATC AACGACTATG AAGAATTCAG CAAAGACTTT GGACAAAGTT 180 TACCAAATGAAAAGCAAACC TCGGGGATAC TGTCTGATCA TCAACAATCA CAATTTTGCA 240 AAAGCACGGGAGAAAGTGCC CAAACTTCAC AGCATTAGGG ACAGGAATGG AACACACTTG 300 GATGCAGGGTTTGAGAATGT TTTTAGCTGG TGGC 334 91 amino acids amino acid single linearprotein 22 Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe IleGlu 1 5 10 15 Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp IleLeu Lys 20 25 30 Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile IleAsn Asp 35 40 45 Tyr Glu Glu Phe Ser Lys Asp Phe Gly Gln Ser Leu Pro AsnGlu Lys 50 55 60 Gln Thr Ser Gly Ile Leu Ser Asp His Gln Gln Ser Gln PheCys Lys 65 70 75 80 Ser Thr Gly Glu Ser Ala Gln Thr Ser Gln His 85 90829 base pairs nucleic acid single linear cDNA 23 ATGGACTTCA GCAGAAATCTTTATGATATT GGGGAACAAC TGGACAGTGA AGATCTGGCC 60 TCCCTCAAGT TCCTGAGCCTGGACTACATT CCGCAAAGGA AGCAAGAACC CATCAAGGAT 120 GCCTTGATGT TATTCCAGAGACTCCAGGAA AAGAGAATGT TGGAGGAAAG CAATCTGTCC 180 TTCCTGAAGG AGCTGCTCTTCCGAATTAAT AGACTGGATT TGCTGATTAC CTACCTAAAC 240 ACTAGAAAGG AGGAGATGGAAAGGGAACTT CAGACACCAG GCAGGGCTCA AATTTCTGCC 300 TACAGGGTCA TGCTCTATCAGATTTCAGAA GAAGTGAGCA GATCAGAATT GAGGTCTTTT 360 AAGTTTCTTT TGCAAGAGGAAATCTCCAAA TGCAAACTGG ATGATGACAT GAACCTGCTG 420 GATATTTTCA TAGAGATGGAGAAGAGGGTC ATCCTGGGAG AAGGAAAGTT GGACATCCTG 480 AAAAGAGTCT GTGCCCAAATCAACAAGAGC CTGCTGAAGA TAATCAACGA CTATGAAGAA 540 TTCAGCAAAG AGAGAAGCAGCAGCCTTGAA GGAAGTCCTG ATGAATTTTC AAATGGGGAG 600 GAGTTGTGTG GGGTAATGACAATCTCGGAC TCTCCAAGAG AACAGGATAG TGAATCACAC 660 ACTTTGGACA AAGTTTACCAAATGAAAAGC AAACCTCGGG GATACTGTCT GATCATCAAC 720 AATCACAATT TTGCAAAAGCACGGGAGAAA GTGCCCAAAC TTCACAGCAT TAGGGACAGG 780 AATGGAACAC ACTTGGATGCAGGGTTTGGG AATGTTTTTA GCTGGTGGC 829 784 base pairs nucleic acid singlelinear cDNA 24 ATGGACTTCA GCAGAAATCT TTATGATATT GGGGAACAAC TGGACAGTGAAGATCTGGCC 60 TCCCTCAAGT TCCTGAGCCT GGACTACATT CCGCAAAGGA AGCAAGAACCCATCAAGGAT 120 GCCTTGATGT TATTCCAGAG ACTCCAGGAA AAGAGAATGT TGGAGGAAAGCAATCTGTCC 180 TTCCTGAAGG AGCTGCTCTT CCGAATTAAT AGACTGGATT TGCTGATTACCTACCTAAAC 240 ACTAGAAAGG AGGAGATGGA AAGGGAACTT CAGACACCAG GCAGGGCTCAAATTTCTGCC 300 TACAGGGTCA TGCTCTATCA GATTTCAGAA GAAGTGAGCA GATCAGAATTGAGGTCTTTT 360 AAGTTTCTTT TGCAAGAGGA AATCTCCAAA TGCAAACTGG ATGATGACATGAACCTGCTG 420 GATATTTTCA TAGAGATGGA GAAGAGGGTC ATCCTGGGAG AAGGAAAGTTGGACATCCTG 480 AAAAGAGTCT GTGCCCAAAT CAACAAGAGC CTGCTGAAGA TAATCAACGACTATGAAGAA 540 TTCAGCAAAG GGGAGGAGTT GTGTGGGGTA ATGACAATCT CGGACTCTCCAAGAGAACAG 600 GATAGTGAAT CACAGACTTT GGACAAAGTT TACCAAATGA AAAGCAAACCTCGGGGATAC 660 TGTCTGATCA TCAACAATCA CAATTTTGCA AAAGCACGGG AGAAAGTGCCCAAACTTCAC 720 AGCATTAGGG ACAGGAATGG AACACACTTG GATGCAGGGT TTGGGAATGTTTTTAGCTGG 780 TGGC 784 261 amino acids amino acid single linear protein25 Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser 1 510 15 Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln 2025 30 Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu 3540 45 Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu 5055 60 Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn 6570 75 80 Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala85 90 95 Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val100 105 110 Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu GluIle 115 120 125 Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp IlePhe Ile 130 135 140 Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys LeuAsp Ile Leu 145 150 155 160 Lys Arg Val Cys Ala Gln Ile Asn Lys Ser LeuLeu Lys Ile Ile Asn 165 170 175 Asp Tyr Glu Glu Phe Ser Lys Gly Glu GluLeu Cys Gly Val Met Thr 180 185 190 Ile Ser Asp Ser Pro Arg Glu Gln AspSer Glu Ser Gln Thr Leu Asp 195 200 205 Lys Val Tyr Gln Met Lys Ser LysPro Arg Gly Tyr Cys Leu Ile Ile 210 215 220 Asn Asn His Asn Phe Ala LysAla Arg Glu Lys Val Pro Lys Leu His 225 230 235 240 Ser Ile Arg Asp ArgAsn Gly Thr His Leu Asp Ala Gly Phe Gly Asn 245 250 255 Val Phe Ser TrpTrp 260 37 base pairs nucleic acid single linear oligonucleotide 26GACTCGAGTC TAGAGTCGAC TTTTTTTTTT TTTTTTT 37 20 base pairs nucleic acidsingle linear oligonucleotide 27 GACTCGAGTC TAGAGTCGAC 20 31 base pairsnucleic acid single linear oligonucleotide 28 GAGGATCCCC AAATGCAAACTGGATGATGA C 31 31 base pairs nucleic acid single linear oligonucleotide29 TTGGATCCAG ATGGACTTCA GCAGAAATCT T 31 277 amino acids amino acidsingle linear protein 30 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys SerIle Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser MetAsp Ser Gly Ile Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro GluMet Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser ThrGly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu ArgGlu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn AspLeu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys GluAsp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser HisGly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp LeuLys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser LeuThr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg GlyThr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175 Gly Val Asp AspAsp Met Ala Cys His Lys Ile Pro Val Asp Ala Asp 180 185 190 Phe Leu TyrAla Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser LysAsp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 GlnTyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu260 265 270 Leu Tyr Phe Tyr His 275 293 amino acids amino acid singlelinear protein 31 Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro AlaGly Gly Glu 1 5 10 15 Glu Asn Met Thr Glu Thr Asp Ala Phe Tyr Lys ArgGlu Met Phe Asp 20 25 30 Pro Ala Glu Lys Tyr Lys Met Asp His Arg Arg ArgGly Ile Ala Leu 35 40 45 Ile Phe Asn His Glu Arg Phe Phe Trp His Leu ThrLeu Pro Glu Arg 50 55 60 Arg Arg Thr Cys Ala Asp Arg Asp Asn Leu Thr ArgArg Phe Ser Asp 65 70 75 80 Leu Gly Phe Glu Val Lys Cys Phe Asn Asp LeuLys Ala Glu Glu Leu 85 90 95 Leu Leu Lys Ile His Glu Val Ser Thr Val SerHis Ala Asp Ala Asp 100 105 110 Cys Phe Val Cys Val Phe Leu Ser His GlyGlu Gly Asn His Ile Tyr 115 120 125 Ala Tyr Asp Ala Lys Ile Glu Ile GlnThr Leu Thr Gly Leu Phe Lys 130 135 140 Gly Asp Lys Cys His Ser Leu ValGly Lys Pro Lys Ile Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly AsnGln His Asp Val Pro Val Ile Pro Leu Asp 165 170 175 Val Val Asp Asn GlnThr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val 180 185 190 Asp Ala Ala SerVal Tyr Thr Leu Pro Ala Gly Ala Asp Phe Leu Met 195 200 205 Cys Tyr SerVal Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn 210 215 220 Gly SerTrp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly 225 230 235 240Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val 245 250255 Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys 260265 270 Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe275 280 285 Phe Pro Lys Ser Asn 290 398 base pairs nucleic acid singlelinear cDNA 32 AAATGCAAAC TGGATGATGA CATGAACCTG CTGGATATTT TCATAGAGATGGAGAAGAGG 60 GTCATCCTGG GAGAAGGAAA GTTGGACATC CTGAAAAGAG TCTGTGCCCAAATCAACAAG 120 AGCCTGCTGA AGATAATCAA CGACTATGAA GAATTCAGCA AAGGGGAGGAGTTGTGTGGG 180 GTAATGACAA TCTCGGACTC TCCAAGAGAA CAGGATAGTG AATCACAGACTTTGGACAAA 240 GTTTACCAAA TGAAAAGCAA ACCTCGGGGA TACTGTCTGA TCATCAACAATCACAATTTT 300 GCAAAAGCAC GGGAGAAAGT GCCCAAACTT CACAGCATTA GGGACAGGAATGGAACACAC 360 TTGGATGCAG GGTTTGAGAA TGTTTTTAGC TGGTGGCA 398 1443 basepairs nucleic acid single linear cDNA 33 CCAAATGCAA ACTGGATGATGACATGAACC TGCTGGATAT TTTCATAGAG ATGGAGAAGA 60 GGGTCATCCT GGGAGAAGGAAAGTTGGACA TCCTGAAAAG AGTCTGTGCC CAAATCAACA 120 AGAGCCTGCT GAAGATAATCAACGACTATG AAGAATTCAG CAAAGACTTT GGACAAAGTT 180 TACCAAATGA AAAGCAAACCTCGGGGATAC TGTCTGATCA TCAACAATCA CAATTTTGCA 240 AAAGCACGGG AGAAAGTGCCCAAACTTCAC AGCATTAGGG ACAGGAATGG AACACACTTG 300 GATGCAGGGG CTTTGACCACGACCTTTGAA GAGCTTCATT TTGAGATCAA GCCCCACGAT 360 GACTGCACAG TAGAGCAAATCTATGAGATT TGGAAAATCT ACCAACTCAT GGACCACAGT 420 AACATGGACT GCTTCATCTGCTGTATCCTC TCCCATGGAG ACAAAGGCAT CATCTATGGC 480 ACTGATGGAC AGGAGGGCCCCATCTATGAG CTGACATCTC AGTTCACTGG TTTGAAGTGC 540 CCTTCCCTTG CTGGAAAACCCAAAGTGTTT TTTATTCAGG CTTGTCAGGG GGATAACTAC 600 CAGAAAGGTA TACCTGTTGAGACTGATTCA GAGGAGCAAC CCTATTTAGA AATGGATTTA 660 TCATCACCTC AAACGAGATATATCCCGGAT GAGGCTGACT TTCTGCTGGG GATGGCCACT 720 GTGAATAACT GTGTTTCCTACCGAAACCCT GCAGAGGGAA CCTGGTACAT CCAGTCACTT 780 TGCCAGAGCC TGAGAGAGCGATGTCCTCGA GGCGATGATA TTCTCACCAT CCTGACTGAA 840 GTGAACTATG AAGTAAGCAACAAGGATGAC AAGAAAAACA TGGGGAAACA GATGCCTCAG 900 CCTACTTTCA CACTAAGAAAAAAACTTGTC TTCCCTTCTG ATTGATGGTG CTATTTTGTT 960 TGTTTTGTTT TGTTTTGTTTTTTTGAGACA GAATCTCGCT CTGTCGCCCA GGCTGGAGTG 1020 CAGTGGCGTG ATCTCGGCTCACCGCGAGCT CCGCCTCCCG GGTTCACGCC ATTCTCCTGC 1080 CTCAGCCTCC CGAGTAGCTGGGACTACAGG GGCCCGCCAT CACACCTGGC TAATTTTTTA 1140 AAAATATTTT TAGTAGAGACAGGGTTTCAC TGTGTTAGCC AGGGTGGTCT TGATCTCCTG 1200 ACCTCGTGAT CCACCCACCTCGGCCTCCCA AAGTGCTGGG ATTACAGGCG TGAGCCACCG 1260 CGCCTGGCCG ATGGTACTATTTAGATATAA CACTATGTTT ATTTACTAAT TTTCTAGATT 1320 TTCTACTTTA TTAATTGTTTTGCACTTTTT TATAAGAGCT AAAGTTAAAT AGGATATTAA 1380 CAACAATAAC ACTGTCTCCTTTCTCTTACG CTTAAGGCTT TGGGAATGTT TTTAGCTGGT 1440 GGC 1443 91 amino acidsamino acid single linear protein 34 Lys Cys Lys Leu Asp Asp Asp Met AsnLeu Leu Asp Ile Phe Ile Glu 1 5 10 15 Met Glu Lys Arg Val Ile Leu GlyGlu Gly Lys Leu Asp Ile Leu Lys 20 25 30 Arg Val Cys Ala Gln Ile Asn LysSer Leu Leu Lys Ile Ile Asn Asp 35 40 45 Tyr Glu Glu Phe Ser Lys Asp PheGly Gln Ser Leu Pro Asn Glu Lys 50 55 60 Gln Thr Ser Gly Ile Leu Ser AspHis Gln Gln Ser Gln Phe Cys Lys 65 70 75 80 Ser Thr Gly Glu Ser Ala GlnThr Ser Gln His 85 90

What is claimed is:
 1. An isolated polypeptide that binds to MORT-1protein, said polypeptide having a sequence comprising: (a) residues1-182 of SEQ ID NO:5; (b) an analog of (a), differing therefrom by nomore than ten changes in the amino acid sequence thereof, each saidchange being a substitution, deletion and/or insertion of a single aminoacid, which analog binds to MORT-1 protein; (c) a fragment of (a) or(b), which fragment binds to MORT-1 protein; or (d) a derivative of (a),(b) or (c) by modification of a functional group which occurs as a sidechain or an N- or C-terminal group of one or more amino acid residuesthereof without changing one amino acid to another of the twentycommonly occurring natural amino acids, which derivative binds to MORT-1protein.
 2. An isolated polypeptide in accordance with claim 1, whereinsaid sequence of part (a) of claim 1 is a MACH protein isoform selectedfrom the group consisting of MACHα1, MACHα2, MACHα3, MACHβ1, MACHβ2,MACHβ3, MACHβ4 and MACHβ5.
 3. An isolated polypeptide in accordance withclaim 1, wherein said sequence of part (a) of claim 1 is MACHα1, MACHβ1or MACHβ3.
 4. An isolated polypeptide in accordance with claim 1,wherein said sequence of part (a) of claim 1 is MACHα1.
 5. An isolatedpolypeptide in accordance with claim 1, wherein said sequence of part(a) of claim 1 is MACHβ1.
 6. An isolated polypeptide in accordance withclaim 1, wherein said sequence of part (a) of claim 1 is MACHβ3.
 7. Anisolated polypeptide having a sequence in accordance with part (a) ofclaim
 1. 8. An isolated polypeptide having a sequence in accordance withpart (b) of claim
 1. 9. An isolated polypeptide having a sequence inaccordance with part (c) of claim
 1. 10. An isolated polypeptide inaccordance with claim 1, wherein said analog of part (b) of claim 1differs from the polypeptide of part (a) by no more than 5 amino acidchanges.
 11. An isolated polypeptide in accordance with claim 1 whereinsaid analog of part (b) of claim 1 differs from the polypeptide of part(a) of claim 1 by no more than 3 amino acid changes.
 12. An isolatedpolypeptide having a sequence comprising a fragment of the polypeptidein accordance with part (a) of claim 1, which fragment binds to MORT-1protein.
 13. A composition comprising a pharmaceutically acceptablecarrier and a polypeptide that binds to MORT-1 protein, whichpolypeptide has a sequence comprising: (a) residues 1-182 of SEQ IDNO:5; (b) an analog of (a), differing therefrom by no more than tenchanges in the amino acid sequence thereof, each said change being asubstitution, deletion and/or insertion of a single amino acid, whichanalog binds to MORT-1 protein; (c) a fragment of (a) or (b), whichfragment binds to MORT-1 protein; or (d) a derivative of (a), (b) or (c)by modification of a functional group which occurs as a side chain or anN- or C-terminal group of one or more amino acid residues thereofwithout changing one amino acid to another of the twenty commonlyoccurring natural amino acids, which derivative binds to MORT-1 protein.14. A composition in accordance with claim 13, wherein said sequence ofpart (a) of claim 13 is a MACH protein isoform selected from the groupconsisting of MACHα1, MACHα2, MACHα3, MACHβ1, MACHβ2, MACHβ3, MACHβ4 andMACHβ5.
 15. A composition in accordance with claim 13, wherein saidsequence of part (a) of claim 13 is MACHα1, MACHβ1 or MACHβ3.
 16. Acomposition in accordance with claim 13, wherein said sequence of part(a) of claim 13 is MACHα1.
 17. A composition in accordance with claim13, wherein said sequence of part (a) of claim 13 is MACHβ1.
 18. Acomposition in accordance with claim 13, wherein said sequence of part(a) of claim 13 is MACHβ3.
 19. A composition in accordance with claim13, wherein said polypeptide has a sequence in accordance with part (a)of claim
 13. 20. A composition in accordance with claim 13, wherein saidpolypeptide has a sequence in accordance with part (b) of claim
 13. 21.A composition in accordance with claim 13, wherein said polypeptide hasa sequence in accordance with part (c) of claim
 13. 22. A composition inaccordance with claim 13, wherein said analog of part (b) of claim 13differs from the polypeptide of part (a) by no more than 5 amino acidchanges.
 23. A composition in accordance with claim 13, wherein saidanalog of part (b) of claim 13 differs from the polypeptide of part (a)by no more than 3 amino acid changes.
 24. A composition in accordancewith claim 13, wherein said polypeptide has a sequence comprising afragment of the polypeptide in accordance with part (a) of claim 13,which fragment binds to MORT-1 protein.